Red Tide Algae Research Articles

Improved use of organic phosphate by Skeletonema costatum through regulation of Zn2+ concentrations.

The maximum growth rate (1.4-2 x 10(5) cells ml(-1) d(-1)), cell final yields (2.6-5.2 x 10(5) cells ml(-1)) and extracellular alkaline phosphatase activity (2.4-10.6 microg phosphate released ml(-1) h(-1)) of the red tide alga, Skeletonema costatum, increased when Zn2+ was increased from 0 to 24 pM, but decreased with 66 pM Zn2+ in growth medium with glycerophosphate as the sole phosphorus source. Extracellular carbonic anhydrase activity and the affinity for HCO3- and CO2 uptake increased when Zn2+ was increased from 0 to 12 pM, but then decreased at higher concentrations. The results suggested that utilization of organic phosphate required more Zn2+ than the uptake of inorganic carbon did, while utilization of dissolved inorganic carbon by Skeletonema costatum was very sensitive to Zn2+ concentration variations.

Growth and photosynthesis limitation of marine red tide alga Skeletonema costatum by low concentrations of Zn2+.

The specific growth rate, cell final yields and extracellar carbonic anhydrase activity of the red tide alga Skeletonema costatum increased with increasing concentrations of Zn2+ from 0 to 12 pM, but decreased when Zn2+ was over 24 pM. However, cells grown under high concentrations of Zn2+ had higher activities of intracellular carbonic anhydrase than those grown under low concentrations of Zn2+. Chlorophyll a-specific light-saturated photosynthetic rate (P(Chla)), dark respiration rate (R(chla)) and apparent photosynthetic efficiency (alpha(chla)) significantly increased with increasing concentrations of Zn2+ from 0 to 3 pM, but decreased when increasing concentrations of Zn2+ from 3 to 66 pM. Photorespiration is the lowest when cells cultured in 3 pM Zn2+. The results suggest physiological activity of Skeletonema costatum is very sensitive to the prevailing concentration of Zn2+.

Some physiological and biochemical changes in marine eukaryotic red tide alga Heterosigma akashiwo during the alleviation from iron limitation

Some physiological and biochemical changes in the marine eukaryotic red tide alga Heterosigma akashiwo (Hada) were investigated during the alleviation from iron limitation. Chlorophyll a/carotenoid ratio increases as a result of iron alleviation. In vivo absorption spectra of iron-limited cells showed a chlorophyll (Chl) absorption peak at 630 nm, 2 nm blue-shifted from the normal position. Low-temperature fluorescence emission spectra of the cells have one prominent Chl emission peak at 685 nm. The cells showed a decrease in fluorescence yield from 685 nm band during alleviation from iron limitation. Low-temperature fluorescence excitation spectra and room-temperature fluorescence spectra indicated an efficient excitation energy transfer in the cells alleviated from iron limitation. Photosynthetic efficiency and carbohydrate content per cell increased after alleviation from iron limitation. Total protein decreased in iron-limited cells, while iron deficiency induced the appearance of specific soluble proteins (17 and 55 kDa).

Response of growth and photosynthesis of marine red tide alga Heterosigma akashiwo to iron and iron stress condition

Heterosigma akashiwo, a red tide alga, was grown in Fe-deficient and Fe-replete batch cultures. Cell final yields and the growth rate were limited when Fe was below 10 nM and alleviated with 100 nM Fe. By comparison with the results under Fe-replete conditions, chlorophyll a-specific and cell-specific light saturated net photosynthetic capacity (Pmchl a and Pmcell), dark respiration rate (Rdchl a and Rdcell) and apparent photosynthetic efficiency (αchl a and αcell) decreased proportionately, whereas the cells became light saturated at higher irradiance under Fe stress (Fe-limited conditions).

Synthesis and stereochemical confirmation of the HI/JK ring system of prymnesins, potent hemolytic and ichthyotoxic glycoside toxins isolated from the red tide alga

Stereocontrolled synthesis of the HI/JK ring model of the prymnesins, glyosidic toxins isolated from the red tide phytoflagellate Prymnesium parvum, is described. Comparison of its 1H and 13C NMR data with those of the natural toxins established the earlier stereochemical assignments.

Fluctuations of the red tide flagellates Chattonella spp. (Raphidophyceae) and the algicidal bacterium Cytophaga sp. in the Seto Inland Sea, Japan

A marine algicidal gliding bacterium Cytophaga sp. strain J18/M01 was isolated in 1990 from a station in northern Harima-Nada, the Seto Inland Sea, Japan, using the harmful red tide alga Chattonella antiqua (Hada) Ono as a susceptible organism. The bacterium can prey upon various species of microalgae. Temporal fluctuations of this bacterium and Chattonella spp. [C. antiqua and C. marina (Subrahmanyan) Hara et Chihara] were investigated weekly at the above station in the summer of 1997 and 1998, using immunofluorescence assay employing highly specific polyclonal antibodies for the bacterium. In the summer of 1997, the cell density of Chattonella spp. showed a maximum value (70 cells ml−1) on 8 July, and decreased thereafter. The bacterium Cytophaga sp. J18/M01 was commonly detected around a few hundreds of cells per milliliter or less. The number of Cytophaga sp. J18/M01 increased after the peak of Chattonella spp., and the maximum cell number of the bacterium was 1350 ml−1. This algicidal bacterium also followed the changes of total amounts of microalgal biomass (chlorophyll a+pheophytin) when Chattonella spp. were absent. In the summer of 1998, Chattonella spp. were relatively less abundant (maximum 21 cells ml−1), and the algicidal bacterium Cytophaga sp. J18/M01 showed a close relationship with the change of total microalgal biomass. The present study suggests that the algicidal bacterium Cytophaga sp. J18/M01 preyed upon, not only harmful red tide microalgae, but also other common microalgae such as diatoms, and the bacterium presumably plays an important role in regulating microalgal biomass in natural marine environments.

Molecular analysis of ribosomal RNA gene of red tide algae obtained from the seto inland sea.

Eleven clones from five species of the planktonic microalgae, (Chattonella antiqua, Chattonella marina, Heterosigma akashiwo, Alexandrium catenella, and Scrippsiella trochoidea), which were collected from the Seto Inland Sea in Japan and from Thailand, were subjected to nucleotide sequence analysis of the D1/D2 domain of the large subunit (LSU) of their ribosomal RNA genes. After amplification by polymerase chain reaction using degenerated primers, whole-nucleotide sequences for the D1/D2 domains of the LSU rRNA gene of 11 microalgae were analyzed. Phylogenic tree analysis using these nucleotide sequences showed each species located in a cluster corresponding to its morphological classification. The nucleotide sequence data for Chattonella spp. suggest that multiple clones of both Chattonella antiqua and Chattonella marina are present in the Seto Inland Sea and that red tide blooms of Chattonella spp. in different years may have contained different clones.

Electrospray ionization tandem mass spectrometry for structural elucidation of protonated brevetoxins in red tide algae.

Brevetoxins, the toxic components of "red tide" algae, all share one of two robust polycyclic ether backbone structures, but they are distinguished by differing side-chain substituents. Electrospray ionization mass spectrometry analyses of brevetoxins have shown that the polyether structure invariably has a very high affinity for sodium cations that results in the production of abundant (M + Na)+ ions even when sodium cations are only present as impurities. Because the ionic charge tends to remain localized on the sodium atom and because at least two bonds must be broken in order to produce polycyclic backbone fragmentation, it is extremely difficult to obtain abundant product ions (other than Na+) from (M + Na)+ brevetoxin precursor ions in low-energy collision-induced dissociation (CID) MS/MS experiments. This report establishes that acid additives (oxalic acid, trifluoroacetic acid, and particularly hydrochloric acid) in aqueous methanol solutions can promote high yields of protonated brevetoxin molecules (MH+ ions) for Btx-1, -2, and -9 brevetoxins. Most importantly, unlike their (M + Na)+ counterparts, MH+ precursor ions offer readily detectable product ions in CID MS/MS experiments, even under low-energy collisions. This direct structural characterization approach has provided decomposition information from brevetoxins that was previously inaccessible, including the identification of diagnostic product ions for "type A" brevetoxins (m/z 611) and "type B" brevetoxins (m/z 779, 473, 179) and characteristic ions for Btx-1 (m/z 221, 139), Btx-2 (m/z 153), and Btx-9 (m/z 157, 85). Precursor ion scans and constant neutral loss scans are proposed to enable screening of individual type A or type B brevetoxins present in naturally occurring mixtures.

Structures and Partial Stereochemical Assignments for Prymnesin-1 and Prymnesin-2: Potent Hemolytic and Ichthyotoxic Glycosides Isolated from the Red Tide Alga Prymnesium parvum

Two glycosidic toxins, prymnesin-1 (PRM1) and prymnesin-2 (PRM2) have been isolated from cultured cells of the red tide phytoflagellate Prymnesium parvum, and the gross structure of PRM2 C96H136Cl3NO35 has been reported in a previous contribution. The molecule possesses unique structural features: a C90 unbranched carbon chain except for a single methyl, five contiguous ether rings (6/6/6/7/6), four distinct 1,6-dioxadecalin units, conjugated double and triple bonds, chlorine and nitrogen atoms, and an uncommon l-xylose. Potent ichthyotoxic and hemolytic properties of the two toxins were also demonstrated. However, the stereochemistry of PRM2 and the structure of PRM1 remained unknown. In the present paper the relative stereochemistry of the polycyclic ether moiety of PRM2 and the structure of PRM1 C107H154Cl3NO44 are reported. Structural elucidation was carried out by extensive analysis of NMR data. Difficulties arising from the poor solubility of the toxins in NMR solvents were overcome by preparing N-...

Biological activities of prymnesin-2 isolated from a red tide alga Prymnesium parvum.

Previously we isolated prymnesin-1 (PRM1) and prymnesin-2 (PRM2) as the major hemolytic and ichthyotoxic agents in the red tide organism Prymnesium parvum and disclosed the structure of PRM2 as a novel glycoside with unusual multiple functionality. PRM2 caused 50% hemolysis of a 1% suspension of dog red blood cells at 0.5 nM. The potency exceeded that of plant saponin by 50000 times. The lethality of PRM2 on freshwater fish Tanichthys albonubes was comparable to that of brevetoxin and also the ichthyotoxicity was markedly enhanced by Ca2+ and by a slight elevation of pH: LC50 in a Ca2+ free medium (pH 7.0) was 300 nM and in the presence of 2 mM Ca2+ (pH 8.0) was 3 nM. The hemolytic activity of PRM2 was not affected by Ca2+ but was markedly affected by blood cell origin. Also, the observation of competitive inhibition by the PRM2 analogues allowed us to assume the presence of a specific binding site on the blood cell surface.

Biological activities of prymnesin-2 isolated from a red tide alga Prymnesium parvum

Previously we isolated prymnesin-1 (PRM1) and prymnesin-2 (PRM2) as the major hemolytic and ichthyotoxic agents in the red tide organism Prymnesium parvum and disclosed the structure of PRM2 as a novel glycoside with unusual multiple functionality. PRM2 caused 50 % hemolysis of a 1 % suspension of dog red blood cells at 0.5 nM. The potency exceeded that of plant saponin by 50 000 times. The lethality of PRM2 on freshwater fish Tanichthys albonubes was comparable to that of brevetoxin and also the ichthyotoxicity was markedly enhanced by Ca2+ and by a slight elevation of pH: LC50 in a Ca2+ free medium (pH 7.0) was 300 nM and in the presence of 2 mM Ca2+ (pH 8.0) was 3 nM. The hemolytic activity of PRM2 was not affected by Ca2+ but was markedly affected by blood cell origin. Also, the observation of competitive inhibition by the PRM2 analogues allowed us to assume the presence of a specific binding site on the blood cell surface. Copyright © 1998 John Wiley & Sons, Ltd.

Application of the random amplified polymorphic DNA (RAPD) technique to distinguishing species of the red tide phytoplankton Chattonella (Raphydophyceae)

The random amplified polymorphic DNA (RAPD) technique was applied to strain and species identification. Four different arbitrary primers were tested with DNAs from seven strains of four species of red tide algae of the genus Chattonella. Two of the primers showed species-dependent profiles, while the other two identified differences between strains. The results indicate that the RAPD technique is useful for distinguishing polymorphisms among species or strains.

Examination of the Conformational Properties of the Prymnesin System : A Computational Chemistry Approach

Abstract Prymnesins are potent ichthyotoxic and hemolytic glycoside molecules isolated from the red tide alga Prymnesium parvum. Their unique molecular structure is conformationally rich with a number of key torsional bonds which were probed with contemporary conformational searching techniques in order to describe the three-dimensional structure of the prymnesin molecule. Important topological features include a backbone twist of 60° and an elongation length of 39.5 Å from ring “A” to ring “N” for the prymnesin-2 molecule in the nOe preferred conformation. An appreciation of these and other molecular structural and energetic features determined by computational techniques were examined with the aim of understanding how prymnesin-2 exhibits it’s potent biological activity in relation to these inherent structural traits.

Virus-like particles in Heterosigma akashiwo (Raphidophyceae): a possible red tide disintegration mechanism

Electron microscopical observations on algal samples collected in 1992 in the middle or final stages of a red tide in Hiroshima Bay, Japan, revealed virus-like particles (VLPs) in the red tide alga Heterosigma akashiwo (Class Raphidophyceae). The host cells appeared moribund and the VLPs were located in and around the nuclear area. The VLPs were icosahedral, ca. 185 nm in diameter, and generated from the periphery of several viroplasms. VLPs were also observed in three other types of H. akashiwo-like cells, which were morphologically distinguishable from each other. The appearance of VLPs in the red tide alga could explain the dramatically rapid termination of this red tide.

Algae Migration Characteristics in Fresh Water Red Tide

To understand the algae migration characteristics in the fresh water red tide, we performed a field survey in the Shorenji Reservoir located in Nabari City, Japan. From the analysis of the field data, it is found that the patterns of vertical distributions of the indices representing biomass are very different in the morning and the afternoon. Since some water quality indices have reverse fluctuations between the surface and the bottom layer in respect of the time series changes and the total biomass of the vertical water column is relatively constant, it is concluded that vertical and daily biomass variation of red tide alga is caused by its daily migration, that is the movement from the bottom layer to the surface in the morning and the reverse movement in the afternoon.

Localization of superoxide anion in the red tide alga Chattonella antiqua.

To investigate the localization of superoxide anion generated in the red tide alga Chattonella antiqua, we added to the incubation medium 3,7-dihydro-6-[4-(2-(N'-(5-fluoresceinyl)thioureido) ethoxy)phenyl]-2-methylimidazo(1,2-a)pyrazin-3-one (FCLA), which is a Cypridina luciferin analogue and a specific fluorescent probe for detecting O2- and 1O2. The live phytoplankter was observed with the laser scanning microscope. At high magnification (x 6400) it was found that the plankter has many large and small verruciform protrusions on the cell surface. When the cell was irradiated with an argon laser (488 nm), a very intense fluorescence was noted at each protrusion. This fluorescence was completely inhibited by the addition of superoxide dismutase to the incubation medium, thus suggesting O2- generation at each verruciform protrusion on the cell surface.

Hydroxyl radical generation by red tide algae

The unicellular marine phytoplankton Chattonella marina is known to have toxic effects against various living marine organisms, especially fishes. However, details of the mechanism of the toxicity of this plankton remain obscure. Here we demonstrate the generation of superoxide and hydroxyl radicals from a red tide unicellular organism, C. marina, by using ESR spectroscopy with the spin traps 5,5-dimethyl-1-pyrroline- N-oxide (DMPO) and N- t-butyl-α-phenylnitrone (PBN), and by using the luminol-enhanced chemiluminescence response. The spin-trapping assay revealed productions of spin adduct of superoxide anion (O 2 −) (DMPO-OOH) and that of hydroxyl radical (· OH) (DMPO-OH) in the algal suspension, which was not observed in the ultrasonic-ruptured suspension. The addition of superoxide dismutase (500 U/ml) almost completely inhibited the formation of both DMPO-OOH and DMPO-OH, and carbon-centered radicals were generated with the disappearance of DMPO-OH after addition of 5% dimethyl sulfoxide (Me 2SO) and 5% ethanol. Furthermore, the generation of methyl and methoxyl radicals, which are thought to be produced by the reaction of hydroxyl radical and Me 2SO under aerobic condition, was identified using spin trapping with a combination of PBN and Me 2SO. Luminol-enhanced chemiluminescence assay also supported the above observations. These results clearly indicate that C. marina generates and releases the superoxide radical followed by the production of hydroxyl radical to the surrounding environment. The velocity of superoxide generation by C. marina was about 100 times faster than that by mammalian phagocytes per cell basis. The generation of oxygen radical is suggested to be a pathogenic principle in the toxication of red tide to susceptible aquaculture fishes and may be directly correlated with the coastal pollution by red tide.

Free radical production by the red tide alga, Chattonella antiqua.

The red tide alga, Chattonella antiqua, was found to show a strong chemiluminescence, using luminol as the reagent, when exposed to ultraviolet irradiation. This luminescence was completely inhibited by ascorbate or catalase, suggesting that hydrogen peroxide was generated by the plankton. Red tide cells exposed to fish gill mucus from young yellowtail resulted in the release of a large number of mucocysts and a weak luminosity, and showed a strong reduction of cytochrome c in the medium. Therefore, the discharge of mucocysts from the red tide, induced by the presence of gill mucus, may be accompanied by the release of active oxygen species. The active oxygen may be involved in depolymerization of mucus glycoproteins from the gill lamellae.

A Monoclonal Antibody Which Recognizes the Cell Surface of Red Tide Alga Gymnodinium nagasakiense.

A monoclonal antibody GN-89 which recognizes the cell surface of Gymnodinium nagasaki-ense G-303-ax2 was obtained. It was reactive with nine strains of Gymnodinium nagasakiense (Gymnodinium Type-'65) and one strain of Gyrodinium sp., but not with Gymnodinium breve, Gymnodinium catenatum, Gymnodinium cf. nagasakiense (Gyrodinium cf. aureolum Hulbert), and some other genera of red tide algae by means of indirect fluorescent immunoassay. The antigen recognized by GN-89 seemed to be very hydrophobic, because GN-89 reacted with the antigen which was dissolved with diethyl ether but not with distilled water. The antigen had a polypeptide moiety, and the antigenic determinant was very heat-stable.

Effects of temperature on the vegetative cell liberation of seven species of red-tide algae from the bottom mud in Osaka Bay

Mud samples were collected from the eastern coast of Osaka Bay from April 1984 to August 1985 and were incubated atin situ mud temperatures to examine the effects of temperature on the liberation of seven species of red-tide algae.