Dinoflagelados (pyrrhophyta) del archipiélago los Roques (Venezuela): familias prorocentraceae y ostreopsidaceae / Dinoflagellates (pyrrhophyta) from the los Roques archipielago (Venezuela): families prorocentraceae and ostreopsidaceae

Se examinaron muestras provenientes del Parque Nacional Archipiélago Los Roques, en el Mar Caribe Sur-Oriental (Venezuela), mediante microscopía óptica. Se describen quince (15) especies de dinoflagelados (Pyrrhophyta) pertenecientes a las familias Procentraceae y Ostreopsidaceae. Los símbolos (*) y (**) señalan nuevos registros para Venezuela y el Mar Caribe respectivamente. Se encontraron diez especies para Prorocetraceae: Mesoporos perforatus (Gran) Lillick, Prorocentrum compressum (Bailey) Abé ex Dodge, *Prorocentrum concavum Fukuyo, *Prorocentrum emarginatum Fukuyo, *Prorocentrum gracile Schütt, Prorocentrum lima (Ehrenberg) Dodge, Prorocentrum micans Ehrenberg, *Prorocentrum rhathymun loeblich III, Sherley & Schmidt, *Prorocentrum scutellum Schröder y Prorocentrum triestinum Schiller. En Ostreopsidaceae se encontraron cinco especies: Coolia monotis Meunier, *Gambierdiscus toxicus Adachi&Fukuyo, ** Ostreopsis lenticularis Schmidt, *Ostreopsis ovata Fukuyo y **Ostreopsis siamensis Schmidt. Finalmente se presenta una clave para las especies Prorocentrum y Ostreopsis encontradas en este estudio.

Analysis of paralytic shellfish poisons by capillary electrophoresis.

A capillary electrophoresis (CE) method with UV detection is described for the separation and determination of underivatized toxins associated with paralytic shellfish poisoning (PSP). Confirmation of the electrophoretic peaks was facilitated by mass spectrometric (MS) detection using an ionspray CE-MS interface and by high-performance liquid chromatography with fluorescence detection. The determination of PSP toxins, such as saxitoxin and neosaxitoxin, in toxic dinoflagellates and scallops is demonstrated and comparisons are made with existing techniques.

Crystallization and preliminary X-ray analysis of a peridinin-chlorophyll a protein from Amphidinium carterae.

Crystals of a water-soluble (Mr approximately 39,000) peridinin-chlorophyll a protein from Amphidinium carterae are reported. The crystals diffract to 2.2 A and belong to a monoclinic (B2) and a triclinic (P1) space group. Spectra of the protein in the crystal and in solution are almost identical.

Presence of saxitoxin in toxic extracts from Gonyaulax polyedra.

A "red tide" bloom of Gonyaulax polyedra occurred in Italy in Autumn, 1988. Algal concentrated extracts and undiluted water samples from the bloom were tested both with the Microtox system and a mouse bioassay, revealing the presence of paralytic shellfish poison-like neurotoxins. Saxitoxin levels evaluated on the basis of toxicological and instrumental analysis showed discrepancies. Other toxins could be present in addition to paralytic shellfish poison.

Florida red-tide toxins (brevetoxins) produce depolarization of airway smooth muscle.

Crude preparations of brevetoxin (PBTX) produce airway contraction; however, it is not known if this toxin-induced mechanical response is coupled to changes in airway smooth muscle membrane potential. Membrane potentials and contractility of in vitro canine trachealis smooth muscle preparations were simultaneously measured with a microelectrode and microforce transducer before and during exposure to either the crude toxin (0.01-1.2 micrograms/ml), or the purified fractions PBTX-2 or PBTX-3 (0.01-0.07 micrograms/ml). Membrane potentials in cultured airway smooth muscle-reaggregate preparations were similarly studied. Toxins produced concentration-dependent depolarizations and contractions in in vitro preparations. These responses were not obtained in the presence of either the muscarinic blocking agent atropine, the sodium channel blocker tetrodotoxin (TTX), 0 mM extracellular Ca2+, or the Ca2+ channel blocker verapamil. The toxins were without effect in cultured cells, whereas acetylcholine produced depolarizations which were blocked in the presence of atropine, but not TTX. This suggested the presence of functional cholinergic receptors in cultured cells, and the PBTX-induced release of endogenous acetylcholine from peripheral nerve endings in the in vitro airway smooth muscle response.

Identification of okadaic acid from a Caribbean dinoflagellate, Prorocentrum concavum.

Lipid-soluble toxins were isolated from a Caribbean strain of the epiphytic dinoflagellate Prorocentrum concavum Fukuyo. The major lipid-soluble toxin (LD50 = 210 +/- 15 micrograms/kg i.p. in mice) was purified by normal and reversed-phase column chromatography and characterized by 1H NMR and mass spectrometry. The toxin was identified as okadaic acid by interpretation of the spectral data. Okadaic acid was previously identified as a toxic component of the related species P. lima (Ehrenberg) Dodge. The finding of okadaic acid production in P. concavum and P. lima, abundant primary producers in the ciguatera-endemic Caribbean, suggests that the role of this toxin in the etiology of ciguatera may be more significant than previously thought.

Toxin composition variations in one isolate of the dinoflagellate Alexandrium fundyense.

A commonly accepted paradigm in the study of saxitoxin-producing dinoflagellates is that the total concentration of all toxins (toxin content) in one isolate can vary with growth conditions, but that the relative abundance of each toxin (toxin composition) does not change. We demonstrate here that dramatic changes in toxin composition do occur in one isolate of Alexandrium fundyense. In nitrogen- and phosphorus-limited semi-continuous cultures, toxin composition varied systematically with growth rate. When cells grew slowly under severe nutrient limitation, toxin composition was dominated by one or at most two toxin epimer pairs; as nutrient stresses eased at higher growth rates, the toxin profiles became more heterogeneous. Steady-state, sustained nitrogen limitation favored the production of toxins C 1,2 and GTX I,IV, whereas phosphorus limitation produced cells with high relative abundance of GTX II,III. STX reached its highest relative abundance when growth was most rapid. The lack of observed compositional changes in most past studies is probably not due to inherent differences in toxin biosynthetic pathways between the strains of Alexandrium examined, but rather to differences in the physiology of cells grown under different culturing modes (batch vs semi-continuous), methods of toxin analysis, and dominant toxins in the particular isolates examined.

Cytotoxic macrolides from a cultured marine dinoflagellate of the genus Amphidinium.

A fourth cytotoxic macrolide, amphidinolide D[1], together with known amphidinolide B[2], has been isolated from a different batch of the cultured dinoflagellate Amphidinium sp., which was symbiotically associated with an Okinawan flatworm Amphiscolops sp. Two-dimensional nmr experiments including 1H-detected heteronuclear multiple-bond correlation (HMBC) resulted in the structure assignment of 1 and structure revision of the diene moiety (C-13-C-15) of amphidinolide B.

Complete amino acid sequence of ferredoxin from Peridinium bipes (Dinophyceae).

The amino acid sequence of the major ferredoxin component isolated from a dinoflagellate, Peridinium bipes, was completely determined. Staphylococcus aureus V8 proteolytic, tryptic and chymotryptic peptides of Cm-ferredoxin were prepared and sequenced. The sequence was Phe-Lys-Val-Thr-Leu-Asp-Thr-Pro-Asp-Gly-Lys-Lys-Ser-Phe-Glu-Cys- Pro-Gly-Asp-Ser-Tyr-Ile-Leu-Asp-Lys-Ala-Glu-Glu-Glu-Gly-Leu-Glu-Leu-Pro- Tyr-Ser - Cys-Arg-Ala-Gly-Ser-Cys-Ser-Ser-Cys-Ala-Gly-Lys-Val-Leu-Thr-Gly-Ser-Ile- Asp-Gln - Ser-Asp-Gln-Ala-Phe-Leu-Asp-Asp-Asp-Gln-Gly-Gly-Asp-Gly-Tyr-Cys-Leu-Thr- Cys-Val - Thr-Tyr-Pro-Thr-Ser-Asp-Val-Thr-Ile-Lys-Thr-His-Cys-Glu-Ser-Glu-Leu. It was composed of 93 amino acid residues with 7 cysteine residues, the highest number found among the chloroplast-type ferredoxins so far sequenced. A cysteine residue was found for the first time at the 89th position in a chloroplast-type ferredoxin. Calculation of the numbers of amino acid differences among chloroplast-type ferredoxins indicates that the Peridinium ferredoxin is far divergent not only from higher plant ferredoxins but also from blue-green algal ferredoxins.

Symbioramide, a novel Ca2+-ATPase activator from the cultured dinoflagellate Symbiodinium sp.

A novel sphingosine derivative, symbioramide, has been isolated from the laboratory-cultured dinoflagellate Symbiodinium sp. as a sarcoplasmic reticulum (SR)Ca2+-ATPase activator, and its structure elucidated to be 1 on the basis of spectral and chemical means.

Some chemical properties of maitotoxin, a putative calcium channel agonist isolated from a marine dinoflagellate.

Maitotoxion, a putative Ca2+ channel agonist, was isolated from cultures of the marine dinoflagellate Gambierdiscus toxicus as a colorless amorphous solid. The toxin reacted positively to Dragendorff's reagent but not to ninhydrin reagent. The mouse lethality of maitotoxin determined by intraperitoneal injection was 0.13 micrograms/kg. Chemical features of the toxin were elucidated mainly by various spectroscopic measurements. The molecular weight of maitotoxin as a disodium salt was estimated to be 3,424.5 +/- 0.5 from the negative fast atom bombardment (FAB) mass spectrum. The presence of two sulfate ester groups in the molecule was apparent from the IR and mass spectra, and from analyses of solvolysis products. Despite of its large size, maitotoxin seems to have no known repeating units, such as amino acids and sugars, no carbonyl groups, no side chains other than methyls or an exomethylenes, and no carbocycles.

Pyrocystis lunula bioluminescence: physicochemical characterization of the luciferin precursor.

The luminescence of the dinoflagellate Pyrocystis lunula is controlled by the reduction state of the luciferin precursor. This molecule (P630) is a chromopeptide more stable than luciferin in methanolic solutions at low temperature. Cations may oxidize P630 or cleave the bond between the peptidic chain and the extended tetrapyrrole. Reduction of P630 is performed enzymatically by a NAD(P)H-dependent oxidoreductase or chemically by 2-mercaptoethanol or dithiothreitol. The state of reduction is monitored by the absorption and fluorescence emission which reveal a conformational change of the chromopeptide depending on the pH. These data will be useful for forthcoming studies on intracellular reducing power regulation and luminescence rhythms of these cells.

In vitro red tide toxin effects on human bronchial smooth muscle.

Airborne Ptychodiscus brevis toxin (PBTX), produced by Ptychodiscus brevis (Florida red tide), induces cough, rhinorrhea, watery eyes, and sneezing in normal individuals and wheezing in subjects with asthma. The mechanism of PBTX-induced contractile response has been investigated by the authors in vitro in dog and rat tissue. PBTX stimulates neuronal sodium channels, resulting in activation of autonomic cholinergic and adrenergic nerve endings in canine upper and lower airway smooth muscle and in rat vas deferens, respectively. This article concerns the investigation of the effect and mechanism of action of PBTX on human airways in order to determine the unique role of the toxin in the pathogenesis of asthma. PBTX elicited contractions of isolated human airway smooth muscle with a threshold concentration of 0.1 micrograms/ml, very similar to values obtained in canine lower airways. Pharmacologic analysis demonstrated that atropine (10(-6) mol/L) blocked the response to both PBTX and acetylcholine; tetrodotoxin (10(-7) mol/L) blocked PBTX but not acetylcholine; and verapamil (10(-5) mol/L) attenuated but neostigmine (10(-8) mol/L) potentiated the response to PBTX. Other selected blockers did not affect the PBTX response. These data indicate that PBTX produces contraction of human lower airway smooth muscle via stimulation of cholinergic nerve fiber sodium channels. The concept that PBTX triggers asthma through this mechanism is strengthened by these results.

The use of fluorochromes in the cytochemical characterization of some phytoflagellates.

Sixteen fluorochromes were tested for the cytochemical characterization of two dinoflagellates (Amphidinuim carterae, Prorocentrum micans) and one chlorophycean flagellate (Dunaliella tertiolecta). Depending on the fluorochrome used, various cellular components (including the plasma membrane, thecal plates, pusule, trichocysts, nucleus, lipid bodies and vacuoles) were revealed. The different colours obtained from single or double fluorochrome staining enabled the differentiation and identification of most cellular components. Protoplasmic staining with Fluorescein diacetate suggested the occurrence of esterases in the three phytoflagellates. Rhodamine B, Neutral Red, FluoroBora P and Nile Blue revealed extensive occurrence of lipoid bodies in A. carterae, but Nile Blue showed considerable difference from the other stains in the inclusion size and intracellular location of these bodies. Chlortetracycline binding, and its inhibition by the Ca2+ionophore A23187, indicated that the plasma membrane, pusule system and trichocysts contain sites of Ca2+ binding. Calcofluor White ST proved superior to Congo Red and Lucifer Yellow in elucidating structural details of the thecal plates of P. micans. While Acridine Orange revealed the presence of surface-coat acidic polysaccharides, the fluoresceinated lectins established their glycoconjugate nature in all the three flagellates. Possible mechanisms of fluorochrome uptake are discussed.

Isolation and spectral characteristics of four toxins from the dinoflagellate Ptychodiscus brevis.

Four ichthyotoxins were isolated from crude toxic extracts of Ptychodiscus brevis using a combination of solvent partitioning, thin layer chromatography and reversed phase high pressure liquid chromatography. The toxins were analyzed by mass, infrared, 1H- and 13C-NMR spectroscopy and were found to constitute two structural families of two toxins each: brevetoxins 1 and 2 and brevetoxins 3 and 4. Comparison with literature data indicates that brevetoxins 1 and 2 are identical to the previously described and characterized 11-ring polyether toxins brevetoxins C and B, respectively. The other two compounds (brevetoxins 3 and 4) also represent a structural pair (with chloroacetone and alpha-methylene-propanal side chains, respectively) which has a different, but related, basic ring-structure.

The chemistry of brevetoxins: a review.

The structure of the unique 'red tide' dinoflagellate neurotoxin, brevetoxin-B is presented and the experimental data supporting the chemical structure is discussed. A brief account of the other brevetoxins and their structural relationships is also presented. A biosynthetic scheme for the natural formation of the brevetoxin skeleton is proposed. Studies of the most toxic of the three pure brevetoxins, brevetoxin-A, indicate a skeleton differing from that of brevetoxin-B.

Acute toxic effects in mice of an extract from the marine algae Gonyaulax monilata.

Unialgal cultures of Gonyaulax monilata were cultured and harvested. A modified Westphall procedure was used to prepare an extract which did not contain saxitoxin, the gonyautoxins and structurally related toxins. The extract was administered i.p. to young adult, male CD-1 mice and produced: sedation, abdominal constriction, fecal clumping in the perianal area, ataxia, tremors, cyanosis, loss of reflexes, convulsions and death (LD50 = 2.28 mg/kg). Gross and microscopic pathology in the treated mice included: acute active hyperemia of the viscera, multifocal areas of necrosis of the musculature of the intestinal wall and diaphragm and the presence of cytoplasmic vacuoles in the peripheral margins of the acinar portion of the pancreas. Clinical pathology of the mice which survived 24 hr included significant elevation in the levels of serum lactic dehydrogenase, glutamic pyruvic and glutamic oxaloacetic transaminases. Some of these mice also had significantly decreased white blood cell counts. The extract administered orally produced similar signs without the abdominal constriction and convulsions (median lethal oral dose = 6.73 mg/kg). Gross pathology findings included extensive and severe congestion of the abdominal visceral organs. Vehicle control mice were normal. In conclusion, G. monilata, previously reported as nontoxic in homeotherms, yields an extract which contains a water soluble glycosidic substance(s) which is lethal to mice.

Occurrence of phosphatidylsulfocholine, the sulfonium analog of phosphatidylcholine in some diatoms and algae.

A survey of seven species of diatoms, one Euglena sp. and one dinoflagellate sp. for the presence of phosphatidylsulfocholine (PSC), the sulfonium analog of phosphatidylcholine (PC), was carried out using 1H-NMR spectroscopy and ammonia desorption chemical ionization mass spectrometry. PSC alone was found only in a non-photosynthetic diatom, Nitzschia alba. PSC, together with PC, was found in four of the diatoms (Nitzschia angularis, Cylindrotheca fusiformis, Phaeodactylum tricornutum and Navicula pelliculosa) in proportions of 6-24% of the total PC + PSC fraction, but little or no PSC (less than 2%) was detected in the remaining two (Cyclotella nana and Navicula incerta). Little or no PSC (less than 2%) was detected in a Euglena sp. by 1H-NMR but its presence was confirmed by 35S-labeling. The amount of PSC, if any, in the dinoflagellate (Amphidinium carterae) was below the level of detection by 1H-NMR.

Effects of highly purified maitotoxin extracted from dinoflagellate Gambierdiscus toxicus on action potential of isolated rat heart.

Maitotoxin is a substance extracted from a dinoflagellate and certain tropical poisonous fish. It is considered as the most potent marine toxin (50 times more potent than tetrodotoxin). In isolated mammalian atria, maitotoxin produces at low doses a positive inotropic effect not modified by reserpine pretreatment and suppressed by Mn2+ ions. In the anaesthetized cat, it induces cardiac arrhythmias and tachycardia leading to cardiac failure. On the other hand, maitotoxin strongly inhibits Na+-K+ ATP-ase from microsomes of cat and human kidneys. In a rat pheochromocytoma cell line, it has been shown to promote a norepinephrine release and an increase in Ca2+ influx. The purpose of this work is to study the effect of maitotoxin on action potential of isolated perfused rat heart to check the existence of a direct effect on the myocardium.