Critical micelle concentrations of allelopathic substances produced by Nannochloris oculata which affect a red tide organism, Gymnodinium breve

Laboratory cultures of the green algae Nannochloris oculata and Nannochloris eucaryotum are known to cause lysis of Gymnodinium breve, which is Florida's red tide organism. Two cytolytic agents were previously identified as methyl palmitate and methyl stearate. In this study, the critical micelle concentrations of these substances were determined by ultraviolet light and turbidimetric methods to be 3.5 +/- 0.3 ppm (methyl stearate) and 4.3 +/- 0.6 (methyl palmitate). There were no significant differences in results obtained using the two methods.     

Uptake of iron by Gomphosphaeria aponina, a possible control organism for the Florida red tide Pytochodiscus brevis.

The assimilation of iron, a growth-limiting metal ion of the cytotoxic marine cyanobacterium, Gomphosphaeria aponina, has been examined in both static and steady-state cultures using 59Fe (III). Uptake of iron by cells followed first-order kinetics, and biphasic (absorption and uptake) behavior was observed as suggested by noted differences between cultures incubated in the light and in the dark. Iron removal in illuminated cultures was rapid, occurring at rates comparable to exponential growth rates. Although uptake was mediated by a chelating agent (EDTA), synthesis and iron assisted transport by hydroxamate-type siderophores was not involved in the uptake of iron by cells, as determined by standard chemical and biological assays of iron deficient cultures. The ecological implications of this research is considered with respect to the cytotoxic antagonism between the cyanobacterium and Florida's red tide organism, Pytochodiscus brevis (Gymnodinium breve).     

The influence of Gomphosphaeria aponina on the growth of Gymnodinium breve and the effect of aponin on the ichthyotoxicity of Gymnodinium breve.

Cells of the marine blue-green alga Gomphosphaeria aponina survive in mixed culture with the marine dinoflagellate, Gymnodinium breve (the Florida red tide organism), but G. breve cells lysed within 4-7 days. It has been established that the cytolytic effect of G. aponina, not nutrient competition, is responsible for the decrease in number of cells. The material elaborated by G. aponina has been termed aponin and has been extracted from the cells. The effect of aponin on the ichthyotoxicity of G. breve cultures was measured using Poecilia sphenops, adapted to sea water, as the assay organism. Aponin is not ichthyotoxic toward P. sphenops, though this material, when incubated with G. breve cultures does destroy the cells and increases the ichthyotoxicity of the cultures. At certain concentrations of aponin, the ichthyotoxicity of G. breve cultures appeared to be mitigate d.     

Growth response of the marine blue-green alga, Gomphosphaeria aponina, to inorganic nutrients and significance to management of Florida red tide.

A bioactive isolate from the blue-green alga Gomphosphaeria aponina is cytolytic towards the dinoflagellate, Gymnodinium breve, Florida's red tide organism. Batch and continuous cultures of G. aponina were used to determine nutrient limitation and to optimize mass-culture conditions. Iron and inorganic carbon were growth limiting; first-order saturation kinetics were observed for both substrates. For Fe3+, kinetic parameters were: Ks = 62 +/- 9 microgram 1(-1), and Ke max = 2.14 days-1. Maximum growth was observed at 150 micrograms Fe3+1(-1), with minimal growth below 10 microgram 1(-1). Cells colonized with increasing Fe3+ supplements, and time to reach maximum culture populations was inversely related to the concentration. For HCO3-,Ks = 62 +/- 4 mg1(-1) and Ke max = 1.3 day-1. Additions of NH4+ up to 200 micrograms 1(-1) were not stimulatory, whereas at 1.0 mg 1(-1) levels, Ke was 50% greater than for NO3- enriched medium. Concentrations greater than 25 micrograms PO4(3-) 1(-1) were stimulatory. However, at 1 mg1(-1), growth was less than in controls. Comparison of similar data available for G. breve would suggest that the inorganic nutrient requirements of G. aponina were minimal. Potential for natural control of G. breve by G. aponina is perhaps related to the efficiency of contact of the two organisms.     

Occurrence of carotenoids and sporopollenin in Nanochlorum eucaryotum, a novel marine alga with unusual characteristics

Pigment analysis of Nanochlorum eucaryotum on two strains grown under different gaseous conditions was performed. Air-gassed control cultures did not differ qualitatively with respect to the content of chlorophylls a and b, carotenes alpha and beta, lutein, violaxanthin, neoxanthin and cryptoxanthin in comparison with cultures grown under natural gas. The absolute pigment content per cell increased in cultures grown with natural gas. Growth of N. eucaryotum depends on CO2 which is present in concentrations up to 2.0 vol% in natural gas. N. eucaryotum cannot utilize methane and is therefore not methylotrophic. In cultures of N. eucaryotum grown with natural gas and in air-gassed cultures under nitrogen deficient conditions the secondary carotenoids canthaxanthin and astaxanthin could be detected. In air-gassed cultures of strain N. eucaryotum Colona the same secondary carotenoids have been found, while secondary carotenoids were never found in strain N. eucaryotum Mainz. Cell walls of N. eucaryotum always contain sporopollenin as confirmed by isolation, elemental analysis, infrared absorption spectrophotometry, acetolysis-resistance and electron microscopy.     

AN EPIZOOTIC OF FLORIDA MANATEES ASSOCIATED WITH A DINOFLAGELLATE BLOOM

Over a 10-wk period in early 1982, 39 Florida manatees ( Trichechus manatus latirostris ) were found dead in the lower Caloosahatchee River and nearby waters of southwestern Florida. Two were killed by boats. The remainder showed no evidence of trauma. Lesions indicative of infectious agents were not identified, and bacteriological and contaminant residue findings were unremarkable. Nonspecific lesions of congestion and hemorrhage were identified in brain tissue. Numerous reports were also received of manatee morbidity. Some distressed manatees showed no biochemical lesions in clinical analyses of blood samples and recovered quickly. Timing of manatee illnesses coincided with fish and double-crested cormorant ( Phalacrocorax auritus ) mortality and morbidity. A widespread bloom of the dinoflagellate red tide organism ( Gymnodinium breve ) also coincided with these incidents. G. breve produces potent neurotoxins (brevetoxins). Circumstantial evidence links these events, and possible routes of exposure may include ingestion of filter-feeding ascidians. Ecological conditions that magnified the extent of the epizootic included an early dispersal of manatees into the area from a nearby winter aggregation site and unusually high salinities that facilitated the inshore spread of the red tide bloom. Management responses to future episodes of red tide in manatee areas are suggested.     

A spectroscopic technique for evaluating sterol-aponin interactions and implications for management of Ptychodiscus brevis red tides

A double-beam quadruple-cell spectrophotometric technique is described that can be used to evaluate the interaction between sterols and HPLC fractions from Nannochloris sp. which are cytolytic (aponin) toward the red tide organism. The interaction with ergosterol but not beta-sitosterol is consistent with complex formation and also with the reduction of cytolytic activity of aponin in the presence of ergosterol, as previously noted. Implications for the mechanism of aponin cytolytic activity are considered.     

Bioassays of APONIN-3 and -4 with rabbit erythrocytes

Rabbit erythrocytes in methanolic phosphate medium were used to bioassay the activity of authentic samples of methyl stearate and methyl palmitate (in 10% methanol:90% water, v/v), which had been identified as apparent oceanic naturally occurring cytolins (APONIN-3 and -4) produced by Nannochloris oculata. The two natural products are notable for cytolytic activity toward the unarmoured dinoflagellate, Gymnodinium breve Davis, an organism responsible for red tides consisting of harmful algal blooms in the Gulf of Mexico and along the eastern coast of the United States. Bioassays were done with heparinized rabbit blood. The absorbance at 540 nm was observed for 15 min in comparison with a sample treated with haemolysing agent. The results indicated that at reasonable concentrations of 1-10 ppm, neither was a haemolysin, although such concentrations caused cytolysis of G. breve cultures.     

Brevenal Is a Natural Inhibitor of Brevetoxin Action in Sodium Channel Receptor Binding Assays

1. Florida red tides produce profound neurotoxicity that is evidenced by massive fish kills, neurotoxic shellfish poisoning, and respiratory distress. Red tides vary in potency, potency that is not totally governed by toxin concentration. The purpose of the study was to understand the variable potency of red tides by evaluating the potential for other natural pharmacological agents which could modulate or otherwise reduce the potency of these lethal environmental events. 2. A synaptosome binding preparation with 3-fold higher specific brevetoxin binding was developed to detect small changes in toxin binding in the presence of potential antagonists. Rodent brain labeled in vitro with tritiated brevetoxin shows high specific binding in the cerebellum as evidenced by autoradiography. Synaptosome binding assays employing cerebellum-derived synaptosomes illustrate 3-fold increased specific binding. 3. A new polyether natural product from Florida's red tide dinoflagellate Karenia brevis, has been isolated and characterized. Brevenal, as the nontoxic natural product is known, competes with tritiated brevetoxin for site 5 associated with the voltage-sensitive sodium channel (VSSC). Brevenal displacement of specific brevetoxin binding is purely competitive in nature. 4. Brevenal, obtained from either laboratory cultures or field collections during a red tide, protects fish from the neurotoxic effects of brevetoxin exposure. 5. Brevenal may serve as a model compound for the development of therapeutics to prevent or reverse intoxication in red tide exposures.     

DIEL PHASING OF THE CELL-CYCLE IN THE FLORIDA RED TIDE DINOFLAGELLATE, GYMNODINIUM BREVE

The diel cycle is a key regulator of the cell-cycle in many dinoflagellates, but the mechanisms by which the diel cycle entrains the cell-cycle remain poorly understood. In this study, we describe diel phasing of the cell-cycle in the Florida red tide dinoflagellate Gymnodinium breve Davis, determine the diel cue which serves to entrain the cell-cycle, and provide evidence for the presence of cyclin-dependent kinase (CDK), a cell-cycle regulator which may be responsive to this cue. Four laboratory isolates from the West Coast of Florida were compared. When grown on a 16:8 h LD cycle, all isolates displayed phased cell division, with the S-phase beginning 6–8 h into the light phase, and mitosis following 12–14 h later, as determined by flow cytometry. A naturally occurring bloom of G. breve, studied over one diel cycle, displayed diel cell-cycle phasing similar to that in the laboratory cultures, with the S-phase beginning during daylight and the peak of mitosis occurring approximately 4 h after sunset. In the laboratory cultures, the dark/light "dawn" transition was found to provide the diel cue which serves to entrain the G. breve cell-cycle, whereas the light/ dark "dusk" transition did not appear to be involved. Evidence for the presence of CDK in G. breve was obtained using two approaches: (1) identification of a 34-kDa protein, immunoreactive to an antibody against a conserved amino acid sequence (α-PSTAIR) unique to the CDK protein family and (2) inhibition of the cell-cycle by olomoucine, a selective CDK inhibitor. Together, these results provide the basis from which one can begin addressing mechanisms by which the diel cycle regulates the cell-cycle in G. breve.     

An epizootic of waterfowl associated with a red tide episode in Florida

During February and March, 1974, an epizootic involving lesser scaup (Aythya affinis) occurred in the Tampa Bay area on the west coast of Florida. Several thousand ducks are estimated to have died. Concurrent with this epizootic was a red tide caused by heavy blooms of the toxic dinoflagellate Gymnodinium breve intoxication were evident in some of the lesser scaup. A controlled experimental feeding of G. breve toxic material to White Pekin ducklings produced illness and death with signs comparable to some of those seen in the scaup.     

CELL CYCLE REGULATORS IN THE FLORIDA RED TIDE DINOFLAGELLATE, GYMNODINIUM BREVE

The diel cycle is a key regulator of the cell cycle in many dinoflagellates, and may play a rate limiting role in bloom formation. Diel phasing of the cell cycle in the Florida red tide dinoflagellate, Gymnodinium breve Davis was previously described in our laboratory. In cultures grown on a 16:8 light:dark cycle, S-phase began 6–8 h into the light phase, and mitosis followed 12–14 h later. The dark/light "dawn" transition was found to provide the diel cue that serves to entrain the G. breve cell cycle. However the cell cycle mechanisms and regulators acted upon by this cue are poorly understood in dinoflagellates. The cell cycle regulatory complex, CDK1-cyclinB, is therefore currently being investigated. Cyclin dependent kinase (CDK) was first identified in G. breve using two approaches: (1) identification of a 34 kDa protein immunoreactive to an antibody raised against a conserved amino acid sequence unique to the CDK protein family (PSTAIR) and (2) inhibition of the cell cycle by olomoucine, a selective CDK inhibitor. Several approaches are currently being employed in order to describe its partner, cyclin B: (1) PCR on genomic DNA with primers deduced from known cyclin box sequences, (2) G. breve expression library screening with an antibody raised against the fission yeast cyclin B (3) western blot analysis on whole protein extracts and cyclin B immunoprecipitated proteins. Current work focuses on the differential expression of the cyclin B homologue in G. breve during its cell cycle and its relation to diel cycle control.     

我国近海赤潮多发区域及其生态学特征

根据有关的研究资料,分析我国近海赤潮多发区分布、主要赤潮生物种类、环境状况及赤潮发生生态学特点.结果表明,我国辽东湾、渤海湾、莱州湾、大连湾、长江口、舟山海域、杭州湾、厦门湾、柘林湾、大鹏湾、珠江口等为赤潮多发区.引发赤潮的因素较多,它与气象、水动力、营养盐及生物环境的变化密切相关,人类活动(如海水养殖、陆源污水排放等)影响加剧近海富营养化是引发赤潮的重要因素;但富营养化并非发生赤潮的唯一条件,低营养海区也可能发生赤潮.目前对诱发赤潮的关键因子及赤潮发生机理,应加强定点长期监测和开展深入研究.     

Isolation and properties of isolated nuclei from the Florida red tide dinoflagellate Gymnodinium breve (Davis)

A simple and rapid method is described for the isolation of nuclei from the Florida red tide dinoflagellate Gymnodinium breve. The nuclei are free of cytoplasmic contamination and are active in endogenous RNA synthesis. The ratio of DNA : RNA : acid-soluble protein : acid-insoluble protein is 1:0.39:0.13:0.63, respectively, and each nucleus contains ca. 113 picograms of DNA. Electrophoretic analysis of the acid-soluble proteins reveals the presence of two histone-like proteins with molecular weights of 12,000 and 13,000.     

氮、磷对小新月菱形藻无机碳利用与碳酸酐酶活性的影响

在实验室条件下研究了氮磷浓度变化对小新月菱形藻无机碳利用与碳酸酐酶活性的影响,结果显示小新月菱形藻随培养液中氮、磷浓度的升高比生长速率明显提高。低氮浓度导致胞外碳酸酐酶活性丧失,但胞内碳酸酐酶活性依然存在。高氮浓度下胞内、外碳酸酐酶活性均明显升高。胞内碳酸酐酶活性在高磷浓度下明显升高,但胞外碳酸酐酶活性并没有受到磷浓度变化的影响。高氮、磷浓度培养下的小新月菱形藻的最大光合作用速率(Vmax)、对CO2亲和力(K0.5(CO2))和光系统II最大光化学效率(Fv/Fm)均明显提高。以上结果表明小新月菱形藻可以通过改变胞内、外碳酸酐酶活性调节无机碳利用以适应不同氮磷浓度的环境。     

Isolation and Properties of Isolated Nuclei from the Florida Red Tide Dinoflagellate Gymnodinium breve (Davis)1

A simple and rapid method is described for the isolation of nuclei from the Florida red tide dinoflagellate Gymnodinium breve. The nuclei are free of cytoplasmic contamination and are active in endogenous RNA synthesis. The ratio of DNA: RNA: acidsoluble protein: acid-insoluble protein is 1:0.39:0.13:0.63, respectively, and each nucleus contains ca. 113 picograms of DNA. Electrophoretic analysis of the acid-soluble proteins reveals the presence of two histone-like proteins with molecular weights of 12,000 and 13,000.     

深圳市污染物排放总量与其东部水体赤潮发生的关系

探讨了深圳市污染物排放总量对深圳东部海域赤潮发生的影响.结果表明：深圳市总氮(TN)、总磷(TP)净排放量以及排污中的N∶P与东部海域甲藻赤潮发生频率呈明显正相关;推算出东部甲藻赤潮爆发的TP、TN年排污阈值分别为3.917×10³ t和2.123×10⁴ t;而近岸排污与硅藻赤潮发生的相关性不明显.举例说明了深圳排污总量控制的具体方式.     

Is inorganic nutrient enrichment a driving force for the formation of red tides?: A case study of the dinoflagellate Scrippsiella trochoidea in an embayment

Red tides (high biomass phytoplankton blooms) have frequently occurred in Hong Kong waters, but most red tides occurred in waters which are not very eutrophic. For example, Port Shelter, a semi-enclosed bay in the northeast of Hong Kong, is one of hot spots for red tides. Concentrations of ambient inorganic nutrients (e.g. N, P), are not high enough to form the high biomass of chlorophyll a (chl a) in a red tide when chl a is converted to its particulate organic nutrient (N) (which should equal the inorganic nutrient, N). When a red tide of the dinoflagellate Scrippsiella trochoidea occurred in the bay, we found that the red tide patch along the shore had a high cell density of 15,000 cells ml⁻¹, and high chl a (56 μg l⁻¹), and pH reached 8.6 at the surface (8.2 at the bottom), indicating active photosynthesis in situ. Ambient inorganic nutrients (NO₃, PO₄, SiO₄, and NH₄) were all low in the waters and deep waters surrounding the red tide patch, suggesting that the nutrients were not high enough to support the high chl a >50 μg l⁻¹ in the red tide. Nutrient addition experiments showed that the addition of all of the inorganic nutrients to a non-red-tide water sample containing low concentrations of Scrippsiella trochoidea did not produce cell density of Scrippsiella trochoidea as high as in the red tide patch, suggesting that nutrients were not an initializing factor for this red tide. During the incubation of the red tide water sample without any nutrient addition, the phytoplankton biomass decreased gradually over 9 days. However, with a N addition, the phytoplankton biomass increased steadily until day 7, which suggested that nitrogen addition was able to sustain the high biomass of the red tide for a week with and without nutrients. In contrast, the red tide in the bay disappeared on the sampling day when the wind direction changed. These results indicated that initiation, maintenance and disappearance of the dinoflagellate Scrippsiella trochoidea red tide in the bay were not directly driven by changes in nutrients. Therefore, how nutrients are linked to the formation of red tides in coastal waters need to be further examined, particularly in relation to dissolved organic nutrients.     

Comparative Toxicity of Gonyaulax monilata and Gymnodinium breve to Annelids, Crustaceans, Molluscs and a Fish

SYNOPSIS. Toxicities of laboratory cultures of the dinoflagellates Gonyaulax monilata and Gymnodinium breve were compared using fish, annelids, crustaceans and molluscs. Toxicity was evaluated as mortality over a 48-hr test period in 100%, 75% 50%, 25% and 10% concentrations of dinoflagellate cultures. Responses of the assay animals indicated that the dinoflagellates do not produce the same toxins. Effects of the toxins seemed uniform within each major group of animals tested. Fish were most sensitive to both G. breve and Gonyaulax monilata; crustaceans were resistant to both; annelids and molluscs were more sensitive to G. monilata than to G. breve.