Heterosigma Carterae Research Articles

PCR-based ribosomal DNA detection technique for microalga (Heterosigma carterae) causing red tide and its application to a biosensor using labeled probe.

A technique for detecting Raphidophycean, a bloom-forming genus of algae, was developed using a specific DNA probe. The design of the probe was based on a sequence polymorphism within the small subunit (SSU) ribosomal RNA gene (rDNA) of this strain by using fluorescence polarization (FP) analysis and the BIAcore 2000 biosensor, which utilized surface plasmon resonance (SPR). The specific sequence in SSU rDNA for Heterosigma carterae was determined by sequence data analysis. One pair of polymerase chain reaction (PCR) probes was designed for use in making the identification. H. carterae SSU rDNA was amplified by PCR. Using a fluoroscein isothiocyanate-labeled or biotin-labeled oligonucleotide probe, the PCR-amplified rDNA was selectively detected as an FP-intensity change via FP analysis or as a resonance-unit change via SPR. Although total time for final detection after sampling was within 3 hours, specific rDNA could be detected within 10 minutes after PCR through these detection methods.

Testing for toxic effects of prey on zooplankton using sole versus mixed diets

Negative effects of prey species on consumers could be due to deterrence, nutritional insufficiency, or toxicity of the prey. These effects can be discerned in experiments in which the suspect prey is offered to the consumers in a food mixture containing another prey item that is not toxic and in which the ingestion rates on the prey and the grazers’ responses (e.g., egg production) are measured. We used this framework to determine whether several algae that have been reported to have harmful effects on grazers (Prorocentrum minimum, low‐and high‐toxin Alexandrium sp., Heterosigma carterae, Thalassiosira rotula, and Phaeodactylum tricornutum) are toxic to females of the calanoid copepod Acartia tonsa. Ingestion, egg production, and egg‐hatching rates were measured for A. tonsa offered sole diets of the suspect alga and mixed diets containing the suspect alga and a control alga (the green flagellate Tetraselmis sp.) at an ecologically relevant concentration (250 µg C L-1) and duration (3 d). With the exception of the Alexandrium strain with the high‐toxin content (16.3 pgSaxitoxin [pgSTX] equivalents cell21), none of the diets studied can be considered toxic. The high‐toxin Alexandrium reduced A. tonsa’s total ingestion rate, and thus egg production, as the proportion of Alexandrium increased in the diet. A. tonsa exhibited significantly reduced ingestion and egg production rates when feeding on sole food diets of H. carterae and P. tricornutum relative to the mixed food diets. However, a comparison of ingestion rates among the diet mixtures revealed that H. carterae and P. tricornutum acted as feeding deterrents when provided as sole foods. These results stress the importance of using mixed food diets when examining putative toxic effects of preys on consumers.

The large capacity for dark nitrate-assimilation in diatoms may overcome nitrate limitation of growth.

• The ability of the diatom Thalassiosira weissflogii to assimilate inorganic N in darkness is compared with that seen in flagellates. • Experiments were conducted with T. weissflogii grown in N-replete and in N-limiting cultures and the rates and capacity for ammonium and nitrate assimilation were determined. • High daily growth rates in the diatom under high-light nitrate-replete conditions are only attainable by continuing nitrate assimilation in darkness using excess C accumulated in the light when nitrate assimilation cannot match C-fixation. The ability to use ammonium in darkness is greater than for nitrate but the ratio of dark to light assimilation for each N source is similar over a wide range of cellular N:C ratios. These capabilities are in strong contrast with those in the flagellates Heterosigma carterae and Heterocapsa illdefina, which are incapable of high nitrate use in darkness. • While the possession of large capacity for dark nitrate-assimilation in diatoms may provide a mechanism that overcomes nitrate limitation of growth, the explanation for the lower capabilities exhibited by flagellates is less clear.

N‐ASSIMILATION IN THE NOXIOUS FLAGELLATE HETEROSIGMA CARTERAE (RAPHIDOPHYCEAE): DEPENDENCE ON LIGHT, N‐SOURCE, AND PHYSIOLOGICAL STATE1

The capabilities of the diel vertically migrating flagellate Heterosigma carterae Hulburt for assimilating ammonium and nitrate into cell‐N in light and in darkness were studied using cells of different N‐status. Ammonium utilization in darkness, except by N‐replete cells, attained>50% of use in the light. However, the capacity to use nitrate was poor in darkness, and less than 20% of nitrate‐N that was taken up in darkness was then actually incorporated into cell‐N. The ability to assimilate N in darkness improved as N‐status (N:C) declined, concurrent with an increasing content of water‐soluble carbohydrate. This carbohydrate was used to support dark N‐assimilation. Cells held in darkness for over a day and that had halted nitrate‐uptake were still capable of taking up ammonium. Furthermore, the act of taking up ammonium appeared to make available a source of C to support nitrate uptake that was previously unavailable. The implications of these results for the ecophysiology of this organism and for the construction of mathematical models of algal growth are considered.

Inhibition of egg hatching success and larvae survival of the scallop, Chlamys farreri, associated with exposure to cells and cell fragments of the dinoflagellate Alexandrium tamarense

We report an apparently novel toxic effect of the dinoflagellate Alexandrium tamarense, manifested by inhibition of the egg hatching success of the scallop, Chlamys farreri. The hatching rate of C. farreri approached only 30% of controls when its fertilised eggs were exposed for 36 h to A. tamarense cells or cellular fragments at a concentration of 100 cells/ml, and the hatching rate was just 5% after exposure to A. tamarense of 500 cells/ml. Similar exposures of the fertilised scallop eggs to two other algal species, the diatom Phaeodactylum tricornutum and the raphidophyte Heterosigma carterae, resulted in no such toxicity or inhibitory effects. Likewise, exposure of eggs to standard STX toxin, as well as to A. tamarense cell contents (supernant of re-suspended algal cells following ultrasonication and centrifugation), did not elicit this inhibitory response. However, exposure of the scallop eggs to cell cultures, intact algal cells, or cell fragments of A. tamarense produced marked toxicity. The alga also influenced larvae at early D-shape stage of scallop. The survival rates began to decrease significantly after exposed for 6 days at concentration of 3000 cells/ml and above; no larvae could survive after 14-day exposure to A. tamarense at 10,000 cells/ml or 20-day at 5000 cells/ml. The results indicated the production of novel substances from A. tamarense which can cause adverse effects on egg hatching and survival of the scallop larvae. The experiment also found that the developmental stages before blastula was the developmental period most sensitive to the A. tamarense toxin(s) and the alga at early exponential stage had the strongest effect on egg hatching comparing with other growth phases. The adverse effect of A. tamarense on early development of scallops may cause decline of shellfish population and may have further impact on marine ecosystem.

CORRIGENDUM

Journal of PhycologyVolume 36, Issue 5 p. 975-975 Free Access CORRIGENDUM First published: 31 January 2003 https://doi.org/10.1046/j.1529-8817.2000.03657.xAboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat The following references were missing from Ingo Maier's book review of Algae by L. E. Graham and L. Wilcox in the August 2000 issue (36:788–9): References Cavalier-Smith, T. & Chao, E. E. 1996. 18S rRNA sequence of Heterosigma carterae (Raphidophyceae), and the phylogeny of heterokont algae (Ochrophyta). Phycologia 35: 500– 10. Clayton, M. 1987. Isogamy and a fucalean type of life history in the Antarctic brown alga Ascoseira mirabilis (Ascoseirales, Phaeophyta). Bot. Mar. 30: 447– 54. Volume36, Issue5October 2000Pages 975-975 ReferencesRelatedInformation

An investigation of non-steady-state algal growth. II. Mathematical modelling of co-nutrient-limited algal growth

The ability of mathematical models to simulate competition for nutrients between three algal species, the diatom <it>Thalassiosira pseudonana</it>, a marine raphidophyte <it>Heterosigma carterae</it> and the dinoflagellate <it>Alexandrium minutum</it>, was investigated. Transient growth models were parameterized and tested using a number of closely controlled laboratory data sets including batch monocultures, batch competition experiments and semi-continuous culture competition experiments. The cell quota model of algal growth was found to be adequate to simulate growth of both the raphidophyte and the dinoflagellate. Batch monoculture data for diatom growth obtained under either nitrogen (N) or silicon (Si) limitation could also be simulated with a quota-style model, which in this case included feedback inhibition of nutrient uptake. However, to simulate both batch and semi-continuous culture experiments (and competition between the species), it was necessary to consider diatom Si-N metabolism. A model was derived which contains a representation of both intracellular N ad Si, and of the interaction of these nutrients within the cell. The model used a co-nutrient limitation based on the perceived functional and structural role of N and Si, respectively, within the cell. Simulations indicated that models capable of adequately representing monoculture growth in batch culture may produce erroneous results when incorporated into models of competition. The co-nutrient model is a first step to producing tractable algal growth models which will represent multiple nutrient stress in transient growth conditions.

An investigation of non-steady-state algal growth. I. An experimental model ecosystem

In order to test rigorously the transient behaviour of mathematical models of algal growth, detailed laboratory data sets with good temporal resolution are required. A series of algal growth experiments was conducted in transient conditions. Monoculture growth of, and competition for nutrients between, three contrasting species of phytoplankton (the diatom<it>Thalassiosira pseudonana</it>, the harmful flagellate<it>Heterosigma carterae</it> and the toxic dinoflagellate<it>Alexandrium minutum</it>) were investigated in different temperature, light and nutrient regimes. Although growth dynamics were qualitatively similar in batch culture, quantitative differences were evident in the growth response of the different species when grown in single yield-limiting nutrient conditions in identical physical conditions. Quantities such as the carbon:nitrogen (C:N) ratio and C and N per cell varied between species and within species under different growth conditions. Such results have particular significance to the development of mathematical models, which commonly represent algal populations as a single homogeneous group using a single currency such as numbers, C or N. Changes in light and temperature regime influenced algal growth:<it>Alexandrium</it> failed to grow at low temperatures, while specific growth rates of <it>Thalassiosira</it> were more sensitive to changes in temperature than those of<it>Heterosigma</it>. Changes in the dominant organism(s) and/or its size or nutrient status may influence the transfer of nutrients within the food web. Commonly, mathematical models make cell growth a function of a single yield-limiting nutrient. Decreased growth rates and high residual nutrient concentrations in competition experiments indicate that this approach is unlikely to be successful in conditions of limited supply of more than one nutrient, where multiple nutrient stresses will be significant.

Highly sensitive chemiluminescence flow-injection detection of the red tide phytoplankton Heterosigma carterae

Two chemiluminescent flow-injection analysis systems for the detection of the red tide Phytoplankton Heterosigma carterae (formerly known as Heterosigma akashiwo) have been developed. In one system, the superoxide (O − 2) released by H. carterae reacts with 2-methyl-6-( p-methoxyphenyl)-3,7-dihydroimidazo[1,2- a]-pyrazin-3-one (MCLA), a superoxide specific probe. In the other system, the hydrogen peroxide released by H. carterae reacts with luminol catalyzed by Arthromyces ramosus peroxidase (ARP). The chemiluminescence is detected by a photomultiplier tube. This system is capable of the rapid determination of H. carterae; the time required for one measurement cycle is ca. 2 min using MCLA-dependant luminescence or 1 min in luminol/ARP luminescence. A linear response was observed from 10 2 to 10 5 cells ml −1 H. carterae. Several other species of phytoplankton gave no response using this system. The detection limit of this method is suitable for detecting H. carterae in the early stage of red tide formation.

Role of associated bacteria in Heterosigma carterae toxicity to salmonids

This paper addresses the role of heterotrophic bacteria in Heterosigma carterae toxicity to salmonids. The experiments performed in this study showed that under the tested conditions, H. carterae is toxic to salmonids only when bacteria were present in the culture. The culture conditions used consisted of: an enriched seawater medium; and either 16L:8D photoperiod or 24 of continuous illumination. When the experiments were performed under these conditions, but in the absence of bacteria, H. carterae was not toxic to salmonids. In the absence of the algae, neither the associated bacteria nor the supernatant of a centrifuged H. carterae culture was toxic to salmonids. The experiments presented here demonstrate that toxicity can be induced in an initially axenic non-toxic H. carterae culture. This toxicity can be achieved through the introduction of the associated bacteria from a toxic H. carterae culture, or by the addition of Vibrio natriegens or Alteromanas haloplanktis (non-toxic marine bacteria). The histological analyses of fish gills suggested that the toxic mechanism might work through the disruption of fish gill lamellae (capillary epithelium separation). Further work is recommended.

Variations in the maximum transport rates for ammonium and nitrate in the prymnesiophyte Emiliania huxleyi and the raphidophyte Heterosigma carterae

Changes in the maximum rates of ammonium and nitrate transport (<it>T</it>max; expressed as C-specific N transport) into<it>Emiliana huxleyi</it> and <it>Heterosigma carterae</it> were related to N status (cellular N:C). There was considerable variation in the magnitude of <it>T</it>max relative to N:C. For nitrate transport in both species and for ammonium in<it>H.carterae</it>, the curve linking N:C and<it>T</it>max was bell shaped; <it>T</it>max increased initially as N:C fell and then <it>T</it>max decreased. In contrast, <it>T</it>max for ammonium in<it>E.huxleyi</it> did not decrease to such low levels and N-specific N transport increased continually as N:C decreased. While<it>T</it>max for ammonium into <it>E.huxleyi</it> could be an order of magnitude higher than the N-transport rate required to support growth, for nitrate transport into both species<it>T</it>max was coupled more closely to growth rate. In<it>H.carterae</it>, <it>T</it>max for ammonium was up to four times that for nitrate. Relationships for ammonium and nitrate<it>T</it>max have been computed for simulations of the growth of <it>E.huxleyi</it> and <it>H.carterae</it>. The implications of these results for modelling ammonium-nitrate interactions are discussed. It is shown that attempts to obtain a relationship between<it>T</it>max and N:C from steady-state chemostat data may result in significant deviations from relationships determined using incubation techniques. Further, it is more important to obtain a good estimate for ammonium <it>T</it>max because of its dominant role in affecting the ammonium-nitrate interaction.

Purification and characterization of phosphoribulokinase from the marine chromophytic alga Heterosigma carterae.

In this study we characterized phosphoribulokinase (PRK, EC 2.7.1. 19) from the eukaryotic marine chromophyte Heterosigma carterae. Serial column chromatography resulted in approximately 300-fold purification of the enzyme. A polypeptide of 53 kD was identified as PRK by sequencing the amino terminus of the protein. This protein represents one of the largest composite monomers identified to date for any PRK. The native holoenzyme demonstrated by flow performance liquid chromatography a molecular mass of 214 +/- 12.6 kD, suggesting a tetrameric structure for this catalyst. Because H. carterae PRK activity was insensitive to NADH but was stimulated by dithiothreitol, it appears that the enzyme may require a thioredoxin/ferredoxin rather than a metabolite mode of regulation. Kinetic analysis of this enzyme demonstrated Michaelis constant values of ribulose-5-phosphate (226 microM) and ATP (208 microM), respectively. In summary, H. carterae PRK is unique with respect to holoenzyme structure and function, and thus may represent an alternative evolutionary pathway in Calvin-cycle kinase development.

The chloroplast bchI gene encodes a subunit of magnesium chelatase in the marine heterokont alga Heterosigma carterae

The bchI gene (synonym: chlI), whose product is involved in magnesium chelatase activity, is located in the small single-copy region of the plastid genome of Heterosigma carterae (heterokont alga, Raphidophyceae; formerly named Olisthodiscus luteus). As a unique feature, bchI in H. carterae overlaps an upstream open reading frame (ORF97) of unknown function by 23 base-pairs. Cells contain mRNAs for the full (1.6 kb) cotranscript as well as transcripts for bchI (1.2 kb) and ORF97 (0.4 kb). Transcription initiation in a plastid run on assay of the ORF97/bchI gene cluster is approximately 5-fold higher in cultures sampled in the light versus the dark. In contrast, Northern analysis shows that all three transcripts are in equal abundance at both light and dark sampling times. An antibody raised against a BchI fragment expressed in Escherichia coli recognized a protein of the expected size in the plastid fraction of H. carterae on a Western blot. Analysis of BchI amino acid sequences suggests that the protein may be membrane translocated and could bind ATP. Phylogenetic analysis of bchI sequences documents a deep evolutionary branching between chlorophyll a/b and non-chlorophyll b plastids.

The chloroplast bchI gene encodes a subunit of magnesium chelatase in the marine heterokont alga Heterosigma carterae

The bchI gene (synonym: chlI), whose product is involved in magnesium chelatase activity, is located in the small single-copy region of the plastid genome of Heterosigma carterae (heterokont alga, Raphidophyceae; formerly named Olisthodiscus luteus). As a unique feature, bchI in H. carterae overlaps an upstream open reading frame (ORF97) of unknown function by 23 base-pairs. Cells contain mRNAs for the full (1.6 kb) cotranscript as well as transcripts for bchI (1.2 kb) and ORF97 (0.4 kb). Transcription initiation in a plastid run on assay of the ORF97/bchI gene cluster is approximately 5-fold higher in cultures sampled in the light versus the dark. In contrast, Northern analysis shows that all three transcripts are in equal abundance at both light and dark sampling times. An antibody raised against a BchI fragment expressed in Escherichia coli recognized a protein of the expected size in the plastid fraction of H. carterae on a Western blot. Analysis of BchI amino acid sequences suggests that the protein may be membrane translocated and could bind ATP. Phylogenetic analysis of bchI sequences documents a deep evolutionary branching between chlorophyll a/b and non-chlorophyll b plastids.

Sulfoquinovosyl diacylglycerols from the alga Heterosigma carterae.

An extract of the chloromonad Heterosigma carterae (Raphidophyceae), cultivated in natural seawater, contained a complex mixture of sulfoquinovosyl diacylglycerols. Palmitoyl (16:0), three isomers of hexadecenoyl (16:1 cis delta 9, delta 11, delta 13), and eicosapentenoyl (20:5) were found to be the main fatty acyl substituents. Exact double-bond sites were determined by mass spectrometry analysis of the corresponding nicotinyl derivatives. Four major sulfoquinovosyl diacylglycerol components were partially purified and identified as 1-4 by interpretation of their nuclear magnetic resonance and mass spectral data. In addition, complete analysis of the H. carterae sulfoquinovosyl diacylglycerols was performed using high-performance liquid chromatography combined with electrospray tandem mass spectrometry.

Photoregulation of Chloroplast Gene Transcription in the Chromophytic Alga Heterosigma carterae.

Light acts as a complex regulator of cellular development and gene expression in photoautotrophs. Although light signals are highly effective in controlling cellular division and chloroplast biogenesis in the toxic marine alga Heterosigma carterae, their influence on gene expression has not been well characterized. To address this need cultures of H. carterae synchronized by an alternating light-dark regime were sampled through 12 h of light and 12 h of dark to characterize cell division, chloroplast complement, and chloroplast RNA abundance. These studies have identified a unimodal pattern of chloroplast transcriptional activity for a suite of cellular and photosynthetic genes. To determine the alga's response to a change in photoperiod, 12-h light/12-h dark-synchronized cultures were transferred to constant light and then periodically sampled. Although cellular and chloroplast division cycles remained synchronized in constant conditions for 24 h, the transcriptional apparatus responded by increasing ctRNA abundance within 45 min of the change in photoperiod. However, the ability of the alga to mount this rapid transcriptional response was limited to the first 2 h of the putative dark period. Thus, the chloroplast transcriptional apparatus of H. carterae may initiate a rapid, temporally gated response to a change in photoperiod that is independent of ongoing light-entrained cellular and chloroplast division cycles.

Vertical distribution of the chloromonad flagellate Heterosigma carterae in columns: implications for bloom development

Blooms of the marine flagellate Heterosigma carterae have been associated with catastrophic fish kills at mariculture facilities around the world. The precise cause(s) of the sudden appearance and disappearance of Heterosigma surface blooms has not been completely described or understood. Environmental data from prior studies of blooms indicate the presence of vertical stratification of the water column that is often induced by freshwater runoff. We report the relatively rapid concentration of Heterosigma cells at the surface of tubes shortly after the addition of distilled water to the surface. This phenomenon of cell concentration in surface waters may partially explain the sudden appearance of Heterosigma cells near the surface and subsequent disappearance when vertical stratification is lost due to turbulence or mixing. The results may lead to bloom mitigation techniques for net pen aquaculture.

Phylogenetic relationships of the Raphidophyceae and Xanthophyceae as inferred from nucleotide sequences of the 18s ribosomal RNA gene

Some earlier studies suggested an evolutionary relationship between the Raphidophyceae (chloromonads) and Xanthophyceae (yellow-green algae), whereas other studies suggested relationships with different algal classes or the &ouml;omycete fungi. To evaluate the relationships, we determined the complete nucleotide sequences of the 18S ribosomal RNA gene from the raphidophytes Vacuolaria virescens, Chattonella subsalsa, and Heterosigma carterae, and the xanthophytes Vaucheria bursata, Botrydium stoloniferum, Botrydiopsis intercedens, and Xanthonema debile. The results showed that the Xanthophyceae were most closely related to the Phaeophyceae. A cladistic analysis of combined data sets (nucleotide sequences, ultrastructure, and pigments) suggested the Raphidophyceae are the sister taxon to the Phaeophyceae-Xanthophyceae clade, but the bootstrap value was low (40%). The raphidophyte genera were united with high (100%) bootstrap values, supporting a hypothesis based upon ultrastructural features that marine and freshwater raphidophytes form a monophyletic group. We examined the relationship between Vaucheria, a siphoneous xanthophyte alga, and the &ouml;omycetes, and we confirmed that Vaucheria is a member of the class Xanthophyceae. Partial nucleotide sequences of the 18S rRNA gene from eight xanthophytes (including Bumillariopsis filiformis, Heterococcus caespitiosus, and Mischococcus sphaerocephalus) produce a phylogeny that is not congruent with the current morphology-based classification scheme.

The fucoxanthin-chlorophyll proteins from a chromophyte alga are part of a large multigene family: structural and evolutionary relationships to other light harvesting antennae.

A fucoxanthin-chlorophyll protein (FCP) cDNA from the raphidophyte Heterosigma carterae encodes a 210-amino acid polypeptide that has similarity to other FCPs and to the chlorophyll a/b-binding proteins (CABs) of terrestrial plants and green algae. The putative transit sequence has characteristics that resemble a signal sequence. The Heterosigma fcp genes are part of a large multigene family which includes members encoding at least two significantly different polypeptides (Fcp1, Fcp2). Comparison of the FCP sequences to the recently determined three-dimensional structure of the pea LHC II complex indicates that many of the key amino acids thought to participate in the binding of chlorophyll and the formation of complex-stabilizing ionic interactions are well conserved. Phylogenetic analyses of sequences of light-harvesting proteins shows that the FCPs of several chromophyte phyla form a natural group separate from the intrinisic peridinin-chlorophyll proteins (iPCPs) of the dinoflagellates: Although the FCP and CAB genes shared a common ancestor, these lineages diverged from each other prior to the separation of the CAB LHC I and LHC II sequences in the green algae and terrestrial plants.

18S rRNA sequence of Heterosigma carterae (Raphidophyceae), and the phylogeny of heterokont algae (Ochrophyta)

Abstract We have sequenced the 18S rRNA gene of the raphidophyte Heterosigma carterae (Hulburt) F.J.R. Taylor and have performed a detailed molecular phylogenetic analysis of heterokont algae. Maximum likelihood, maximum parsimony and two distance methods (Fitch and neighbor joining) all show that Heterosigma is specifically related to eustigmatophytes and chrysophytes: somewhat more distant are the xanthophytes and brown algae, recently classified together in a superclass Fucistia. Our trees also show that chloroplasts have been lost twice independently in pedinellids. We discuss the phylogeny and nomenclature of heterokont algae (phylum Ochrophyta or Ochrista), and present a revised phylogenetic classification of Ochrophyta down to the level of subclass. We transfer both raphidophytes and eustigmatophytes into the infraphylum Chrysista (which previously comprised only Fucistia and chrysophytes), even though they lack an intraplastid eyespot and associated swelling on the smooth cilium that characterize ...