Relationships between dynamics of red tide-causing raphidophycean flagellates and algicidal micro-organisms in the coastal sea of Japan

Temporal fluctuations of algicidal micro-organisms against the red tide causing raphidophycean flagellates Chattonella antiqua (Hada) Ono and Heterosigma akashiwo (Hada) Hada ex Hara et Chihara were investigated using the microplate most probable number (MPN) method in northern Hiroshima Bay and Harima-Nada, the Seto Inland Sea, in 1992 and 1993. In Har-ima-Nada, both flagellates appeared at low levels (< 1 cell mL^?1), and killer micro-organisms against the two flagellates (C-killer for C. antiqua and H-killer for H. akashiwo) also appeared at low densities (< 2 mL^?1). In northern Hiroshima Bay, C. antiqua cells were scarce (< 1 cell mL^?1), and C-killers occurred at a low level (≤ 3.4 mL^?1). Conversely, red tides of H. akashiwo occurred there in June of both years. The dynamics of H-killers revealed a close relationship with that of H. akashiwo populations. H-killers followed the increase of H. akashiwo cells, reached a maximum level after the beginning of decline of H. akashiwo, maintained a high level for at least 1 week after the crash of bloom, and then decreased. C-killers consistently remained at low densities during the period of H. akashiwo red tides in both 1992 and 1993. Hence, algicidal micro-organisms specifically associated with the occurrence and crash of H. akashiwo red tides, and presumably contributed to the rapid termination of the red tides in the coastal seas such as northern Hiroshima Bay.     

Cyst morphology of a chain-forming unarmored dinoflagellate Gyrodinium impudicum Fraga et Bravo

Cysts of a chain‐forming dinoflagellate Gyrodinium impudicum Fraga et Bravo (Gymnodiniales) were found in surface sediments of Harima‐Nada and Nakaumi, western Japan. The detailed morphology of living and empty cysts is described. The living cysts are roundish to ellipsoidal in polar view, and hemispherical in lateral view. Among three empty cysts obtained, two different archeopyles were observed; either a long slit with an operculum, or a hole with irregular zigzag outline. The living cysts of Gyro. impudicum are morphologically similar to those of the genus Chattonella antiqua (Hada) Ono and Chattonella marina (Subrahmanyan) Y. Hara et Chihara (Raphidophyceae), except cyst color and contents. The living cysts of Gyro. impudicum were rarely encountered, and their density was always less than 1 cell in 1 cm^?3 in the present samples.     

Broad Salinity Tolerance as a Refuge from Predation in the Harmful Raphidophyte Alga Heterosigma akashiwo (Raphidophyceae)

The ability of harmful algal species to form dense, nearly monospecific blooms remains an ecological and evolutionary puzzle. We hypothesized that predation interacts with estuarine salinity gradients to promote blooms of Heterosigma akashiwo (Y. Hada) Y. Hada ex Y. Hara et M. Chihara, a cosmopolitan toxic raphidophyte. Specifically, H. akashiwo's broad salinity tolerance appears to provide a refuge from predation that enhances the net growth of H. akashiwo populations through several mechanisms. (1) Contrasting salinity tolerance of predators and prey. Estuarine H. akashiwo isolates from the west coast of North America grew rapidly at salinities as low as six, and distributed throughout experimental salinity gradients to salinities as low as three. In contrast, survival of most protistan predator species was restricted to salinities >15. (2) H. akashiwo physiological and behavioral plasticity. Acclimation to low salinity enhanced H. akashiwo's ability to accumulate and grow in low salinity waters. In addition, the presence of a ciliate predator altered H. akashiwo swimming behavior, promoting accumulation in low‐salinity surface layers inhospitable to the ciliate. (3) Negative effects of low salinity on predation processes. Ciliate predation rates decreased sharply at salinities <25 and, for one species, H. akashiwo toxicity increased at low salinities. Taken together, these behaviors and responses imply that blooms can readily initiate in low salinity waters where H. akashiwo would experience decreased predation pressure while maintaining near‐maximal growth rates. The salinity structure of a typical estuary would provide this HAB species a unique refuge from predation. Broad salinity tolerance in raphidophytes may have evolved in part as a response to selective pressures associated with predation.     

Morphological and genetic characterization of bloom‐forming Raphidophyceae from Brazilian coast

Massive fish kills caused by bloom‐forming species of the Raphidophyceae occur in many marine coastal areas and often cause significant economic losses. The ultrastructure and phylogeny of marine raphidophytes from the Brazilian coast have not been fully analyzed. Here, we present the first combined morphological and genetic characterization of raphidophyte strains from the Brazilian coast. Ten strains of four raphidophyte species (Chattonella subsalsa, C. antiqua, Heterosigma akashiwo, and Fibrocapsa japonica) were characterized based on morphology (including ultrastructure) and LSU rDNA sequences. Chattonella subsalsa and C. antiqua formed two distinct genetic clades. We found that the cell size is the only phenotypic feature separating C. subsalsa and C. antiqua strains from Brazil, whereas traditional characteristics used for species separation in the genus Chattonella (i.e., tail size, chloroplast presence in the tail, ‘oboe‐shaped’ mucocysts, and presence of thylakoids in the pyrenoid matrix) were not sufficiently discriminative, due to their overlapping in the two taxa. The phylogenetic analysis indicated intra‐specific geographic differences among C. subsalsa sequences, with two subclades: one formed by isolates from Brazil, USA, and Iran, and another by a sequence from the Adriatic Sea (Italy). Fibrocapsa japonica also showed intra‐specific geographic differences, with a sequence from a Brazilian strain grouped with strains from Japan, Australia, and Germany, all of them distinct from the Italian isolates. This is the first combined morphological and phylogenetic analysis of raphidophytes from the South Atlantic. Our findings broaden knowledge of the biodiversity of this important bloom‐forming algal group.     

INTERSTRAIN VARIABILITY IN PHYSIOLOGY AND GENETICS OF HETEROSIGMA AKASHIWO (RAPHIDOPHYCEAE) FROM THE WEST COAST OF NORTH AMERICA1

High levels of intraspecific variability are often associated with HAB species, and this variability is likely an important factor in their competitive success. Heterosigma akashiwo (Hada) Hada ex Y. Hara et M. Chihara is an ichthyotoxic raphidophyte capable of forming dense surface‐water blooms in temperate coastal regions throughout the world. We isolated four strains of H. akashiwo from fish‐killing northern Puget Sound blooms in 2006 and 2007. By assessing numerous aspects of biochemistry, physiology, and toxicity, we were able to describe distinct ecotypes that may be related to isolation location, source population, or bloom timing. Contrasting elements among strains were cell size, maximum growth and photosynthesis rates, tolerance of low salinities, amino acid use, and toxicity to the ciliate grazer Strombidinopsis acuminatum (Fauré‐Fremiet). In addition, the rDNA sequences and chloroplast genome of each isolate were examined, and while all rDNA sequences were identical, the chloroplast genome identified differences among the strains that tracked differences in ecotype. H. akashiwo strain 07A, which was isolated from an unusual spring bloom, had a significantly higher maximum potential photosynthesis rate (28.7 pg C · cell^?1 · h^?1) and consistently exhibited the highest growth rates. Strains 06A and 06B were not genetically distinct from one another and were able to grow on the amino acids glutamine and alanine, while the other two strains could not. Strain 07B, which is genetically distinct from the other three strains, exhibited the only nontoxic effect. Thus, molecular tools may support identification, tracking, and prediction of strains and/or ecotypes using distinctive chloroplast gene signatures.     

Assessment of EvaGreen-based quantitative real-time PCR assay for enumeration of the microalgae Heterosigma and Chattonella (Raphidophyceae)

Heterosigma akashiwo and Chattonella species (Raphidophyceae) are difficult to detect and quantify in environmental samples because of their pleomorphic and fragile cell nature. In this study, we developed a quantitative real-time polymerase chain reaction (qRT-PCR) assay for the enumeration of these algal taxa using a new DNA-binding dye, EvaGreen. Species-specific qRT PCR primers to H. akashiwo, Chattonella antiqua, Chattonella marina, Chattonella ovata, and Chattonella subsalsa were designed to target the ITS2 rRNA gene intergenic region. Primer specificities were tested via BLAST searches. In addition, specificity was verified using empirical tests, including competitive PCR. The qRT PCR assay analyzing C _t value and the log of cell number showed a significant linear relationship (r ²?≥?0.997). When light microscopy was used to monitor the population dynamics of targeted Raphidophyceae from Lake Shihwa, H. akashiwo was detected in ten samples and no Chattonella spp. were detected (70 samples collected from May, 2007 to January, 2008). In contrast, when the qRT-PCR assay was used, H. akashiwo was detected in 41 samples. C. antiqua, C. marina, and C. ovata were detected in eight samples. Most of the samples analyzed using qRT-PCR assays showed higher algal numbers than did those assayed via microscopy, suggesting that the enumeration of Raphidophyceae via classic microscopic methods most likely underestimates true algal concentration.     

CRYPTIC DIVERSITY IN PHYTOPLANKTON CULTURES IS REVEALED USING A SIMPLE PLATING TECHNIQUE1

A simple technique has been developed to probe the occurrence of cryptic genetic diversity in populations of laboratory‐maintained phytoplankton cultures. This agarose‐based method allows the investigator to plate a broad range of fragile algae, including representatives of the Raphidophyceae, Synurophyceae, Prymnesiophyceae, and Dinophyceae. Heterosigma akashiwo (Hada) Hada ex Y. Hara et Chihara was selected as a model system for our genetic diversity study. Further optimization of the plating technique for this alga demonstrated that colony formation was independent of the physiological state of the parent culture and yet dependent on incident light intensity. The density at which cells were plated affected colony formation and the rate of growth, with intermediate densities (～10³ cells per plate) performing best. High‐metal stress was used as the selective screen for assessing genetic variability within a single H. akashiwo culture. To this end, “clonal” lines (quotation marks used to indicate that within‐clone diversity was expected) were generated from individual plated colonies, and their tolerance was measured by plating on selective medium. Results of these experiments suggest the following: (i) “clonal” lines generated from a single H. akashiwo culture displayed a significant variation in stress tolerance; (ii) “clonal” lines chosen for their ability to grow on selective plates retained this tolerance in the absence of stress, indicating that the observed variation is heritable; and (iii) genetic variation is continually generated in growing cultures. Our results are consistent with a conceptual model, presented here, in which stress tolerance among the individuals in a culture has a genetic basis that varies over a continuous spectrum.     

The photobiology of Heterosigma akashiwo. Photoacclimation,diurnal periodicity,and its ability to rapidly exploit exposure to high light

Periodic and seasonal exposure to high light is a common occurrence for many near‐shore and estuarine phytoplankton. Rapid acclimatization to shifts in light may provide an axis by which some species of phytoplankton can outcompete other microalgae. Patterns of photoacclimation and photosynthetic capacity in the raphidophyte Heterosigma akashiwo (Hada) Hada ex Hara et Chihara isolated from the mid‐Atlantic of the United States were followed in continuous cultures at low‐ and high‐light intensities, followed by reciprocal shifts to the opposite light level. The maximum quantum yield (F_v/F_m) as well as the photosynthetic cross‐section (σ_PSII) of photosystem II was higher in high‐light cultures compared to low‐light cultures. Significant diurnal variability in photochemistry and photoprotection was noted at both light levels, and high‐light‐acclimated cultures displayed greater variability in photoprotective pathways. When shifted from low to high light, there was only a slight and temporary decline in maximum quantum yield, while cell specific growth more than doubled within 24 h. Rapid acclimation to high light was facilitated by short‐term photoprotection (nonphotochemical quenching), reduced PSII reaction center connectivity, and electron transport. Short‐term increases in de‐epoxidated xanthophyll pigments contributed to nonphotochemical protection, but lagged behind initial increases in nonphotochemical quenching and were not the primary pathway of photoprotection in this alga. By 48 h, photochemistry of cultures shifted from low to high light resembled long‐term high‐light‐acclimated cultures. This isolate of H. akashiwo appears well poised to exploit rapid shifts in light by using unique cellular adjustments in light harvesting and photochemistry.     

Assessment of Microzooplankton Grazing on <Emphasis Type="Italic">Heterosigma akashiwo</Emphasis> Using a Species- Specific Approach Combining Quantitative Real-Time PCR (QPCR) and Dilution Methods

Delaware’s Inland Bays (DIB) are subject to numerous mixed blooms of harmful raphidophytes each year, and Heterosigma akashiwo is one of the consistently occurring species. Often, Chattonella subsalsa, C. cf. verruculosa, and Fibrocapsa japonica co-occur with H. akashiwo, indicating a dynamic consortium of raphidophyte species. In this study, microzooplankton grazing pressure was assessed as a top–down control mechanism on H. akashiwo populations in mixed communities. Quantitative real-time polymerase chain reaction (QPCR) with species-specific primers and probes were used in conjunction with the dilution method to assess grazing pressure on H. akashiwo and other raphidophytes. As a comparison, we measured changes in chlorophyll a (chl a) to determine whole community growth and mortality caused by grazing. We detected grazing on H. akashiwo using QPCR in samples where chl a analyses indicated little or no grazing on the total phytoplankton community. Overall, specific microzooplankton grazing pressure on H. akashiwo ranged from 0.88 to 1.88 day⁻¹ at various sites. Experiments conducted on larger sympatric raphidophytes (C. subsalsa, C. cf. verruculosa and F. japonica) demonstrated no significant microzooplankton grazing on these species. Grazing pressure on H. akashiwo may provide a competitive advantage to other raphidophytes such as Chattonella spp. that are too large to be consumed at high rates by microzooplankton and help to shape the dynamics of this harmful algal bloom consortium. Our results show that QPCR can be used in conjunction with the dilution method for evaluation of microzooplankton grazing pressure on specific phytoplankton species within a mixed community. An erratum to this article can be found at     

RAPHIDOPHYCEAE [CHADEFAUD EX SILVA] SYSTEMATICS AND RAPID IDENTIFICATION: SEQUENCE ANALYSES AND REAL‐TIME PCR ASSAYS1

Species within the class Raphidophyceae were associated with fish kill events in Japanese, European, Canadian, and U.S. coastal waters. Fish mortality was attributable to gill damage with exposure to reactive oxygen species (peroxide, superoxide, and hydroxide radicals), neurotoxins, physical clogging, and hemolytic substances. Morphological identification of these organisms in environmental water samples is difficult, particularly when fixatives are used. Because of this difficulty and the continued global emergence of these species in coastal estuarine waters, we initiated the development and validation of a suite of real‐time polymerase chain reaction (PCR) assays. Sequencing was used to generate complete data sets for nuclear encoded small‐subunit ribosomal RNA (SSU rRNA; 18S); internal transcribed spacers 1 and 2, 5.8S; and plastid encoded SSU rRNA (16S) for confirmed raphidophyte cultures from various geographic locations. Sequences for several Chattonella species (C. antiqua, C. marina, C. ovata, C. subsalsa, and C. verruculosa), Heterosigma akashiwo, and Fibrocapsa japonica were generated and used to design rapid and specific PCR assays for several species including C. verruculosa Hara et Chihara, C. subsalsa Biecheler, the complex comprised of C. marina Hara et Chihara, C. antiqua Ono and C. ovata, H. akashiwo Ono, and F. japonica Toriumi et Takano using appropriate loci. With this comprehensive data set, we were also able to perform phylogenetic analyses to determine the relationship between these species.     

Extracellular organics from specific cultures of Heterosigma akashiwo (Raphidophyceae) irreversibly alter respiratory activity in mammalian cells

Raphidophyte blooms have been well documented in several coastal areas around the world. Centring raphidophyte-bloom research has been a focus evolving around issues of ichthyotoxicity, allelopathy and anti-predatory activity. However, the details of these phenomena such as the identity of the compounds and the mechanisms underlying these processes are poorly understood. One such raphidophyte, Heterosigma akashiwo (Hada) Hara et Chihara, has historically received much attention with regard to its ichthyotoxic and allelopathic properties. In this study, we collected extracellular organic compounds from cultures of nine H. akashiwo isolates and tested those exudates on two mammalian cell lines: rat osteoblastic sarcoma (UMR-106) and human embryonic kidney (HEK-293). A tetrazolium colourimetric assay was used to determine the activity of mitochondrial dehydrogenases. Exposure of the mammalian cell lines to exudates collected from cultures of H. akashiwo (strain 764) significantly increased activity in a concentration- and time-dependent manner. Exudate concentrations of as little as 0.3 mg ml⁻¹ elicited a stimulatory response in the mammalian cells. This is comparable to the range of exudate concentrations that were originally in the algal cultures (>0.1 mg ml⁻¹). Significant increases in activity were observed 12–24 h following continuous or 1 h (transient) exposure to the exudate. Production of the stimulatory bioactive exudate was not altered by nutrient-stressed H. akashiwo cultures (reduced iron, phosphate or nitrate). Collectively, these bioactive compound(s) consistently increased cellular activity 3–15-fold. Interestingly, of the nine isolates tested, four of them produced the stimulatory exudate, whereas four others did not produce the stimulatory compound(s) and isolate 560R produced a compound(s) that was inhibitory in nature. Thus, we have shown that cultures of H. akashiwo produce organic compounds that can alter the metabolic activity of mammalian cells. Future isolation and characterization of these bioactive compounds may determine them to have ecological relevance, potentially involved in the ichthyotoxic, allelopathic and/or anti-predatory behaviour of this alga.     

Chemotaxonomic survey of sterols and fatty acids in six marine raphidophyte algae

Fatty acid and sterol profiles allowed for clear discrimination betweentheraphidophyte genera Chattonella,Heterosigma, Fibrocapsa andOlisthodiscus, but exhibited little differentiation forindividual Chattonella species(C.marina, C. antiqua and C.subsalsa). Sterol and fatty acid profiles do not support theseparation of Chattonella antiqua and C.marina as distinct species. Ecophenotypic variations in lipidprofiles were also observed between Chattonella strainsfromdifferent geographic locations. Sterol signatures which may be useful aschemotaxonomic markers were: the absence of C₂₇ sterols (cholesteroland 24-dihydrozymosterol) in Heterosigma akashiwo; thepresence of isofucosterol in Chattonella; and theoccurrence of brassicasterol, poriferasterol and fucosterol inOlisthodiscus luteus. High levels of eicosapentaenoic acid(EPA; 17-27% of fatty acids) were present in all raphidophyte species. Lipidcomposition correlated more closely to recent molecular classification ofraphidophytes than carotenoid pigments.     

CHARACTERIZATION OF HaRNAV,A SINGLE‐STRANDED RNA VIRUS CAUSING LYSIS OF HETEROSIGMA AKASHIWO (RAPHIDOPHYCEAE)1

HaRNAV, a novel virus that infects the toxic bloom‐forming alga Heterosigma akashiwo (Hada) Hada ex Hada et Chihara, was characterized based on morphology, pathology, nucleic acid type, structural proteins, and the range of host strains that it infects. HaRNAV is a 25‐nm single‐stranded RNA (ssRNA) virus with a genome size of approximately 9100 nucleotides. This is the first report of an ssRNA virus that causes lysis of a phytoplankton species. The virus particle is sensitive to chloroform and contains at least five structural proteins ranging in apparent size from 24 to 34 kDa. HaRNAV infection causes swelling of the endoplasmic reticulum and progeny virus particles assemble in the cytoplasm of the host, frequently in crystalline arrays. The infectivity of HaRNAV was tested against 15 strains of H. akashiwo isolated from Japanese waters, the Northeast Pacific, and the Northwest Atlantic. HaRNAV caused lysis of three strains from the Northeast Pacific and two strains from Japan but none from the Northwest Atlantic. The characterization of HaRNAV demonstrates that HaRNAV is a novel type of phytoplankton virus but has some similarities with plant viruses belonging to the Sequiviridae and to other known ssRNA viruses. Further genomic analysis, however, is necessary to determine any phylogenetic relationships. The discovery of HaRNAV emphasizes the diversity of H. akashiwo viral pathogens and, more importantly, algal–virus pathogens and the complexity of virus–host interactions in the environment.     

HETEROSIGMA AKASHIWO (RAPHIDOPHYCEAE) RESTING CELL FORMATION IN BATCH CULTURE: STRAIN IDENTITY VERSUS PHYSIOLOGICAL RESPONSE 1,2

In this study, we examined the impact of environmental perturbation on the movement of the toxic bloom‐forming alga Heterosigma akashiwo (Hada) Hada ex Y. Hara et Chihara [syn. H. carterae (Hulburt) F.J.R. Taylor] between vegetative and resting cell phases of the life history. Resting state induction, in batch culture, was most effective when vegetative cells were subjected to low temperature (10° C) and darkness for extended time periods. Heterosigma cells in stasis had neither a cell wall nor scales but were surrounded by a calyx, most probably of polysaccharide composition. The resting cell was completely immobile, although both flagella remained attached. Heterosigma resting cells did not require a maturation period before successful activation to the vegetative state could occur. Cell division and motility were impacted sequentially during both the induction and activation phases of resting cell development. Our data show that Heterosigma had an obligate light requirement for resting cell activation. In replete medium, very low light fluences of 5 μmol photons·m ^{? 2}·s ^{? 1} were as effective as 60 μmol photons·m ^{? 2}·s ^{? 1} in the initiation of activation. Such sensitivity to extremely low light might give Heterosigma a competitive advantage for bloom formation in nature. Reduced nitrate levels significantly shortened the temporal transition of vegetative cells into the resting cell phase of the life history. Additionally, when resting cells induced in nitrate‐limited medium were activated under nitrate‐replete condition, the efficiency of the activation response was directly correlated to light availability. Both vegetative and resting cells maintained a haploid DNA complement. Rapid amplified polymorphic DNA (RAPD) analysis demonstrated variation in genetic identity among axenic Heterosigma strains. Strain identity influenced success in resting cell induction and survival in stasis. To date, no defined sexual cycle has been described. These observations are discussed in terms of population fitness. The data presented in this report provide a model algal system wherein the molecular events that govern long‐term stasis in an obligately autotrophic organism can now be assessed.     

Monoamine-dependent Production of Reactive Oxygen Species Catalyzed by Pseudoperoxidase Activity of Human Hemoglobin

Hemoglobin (Hb) solution-based blood substitutes are being developed as oxygen-carrying agents for the prevention of ischemic tissue damage and low blood volume-shock. However, the cell-free Hb molecule has intrinsic toxicity to the tissue since harmful reactive oxygen species (ROS) are readily produced during autoxidation of Hb from the ferrous state to the ferric state, and the cell-free Hb also causes distortion in the oxidant/antioxidant balance in the tissues. There may be further hindering dangers in the use of free Hb as a blood substitute. It has been reported that Hb has peroxidase-like activity oxidizing peroxidase substrates such as aromatic amines. Here we observed the Hb-catalyzed ROS production coupled to oxidation of a neurotransmitter precursor, β-phenylethylamine (PEA). Addition of PEA to Hb solution resulted in generation of superoxide anion (O₂^??). We also observed that PEA increases the Hb-catalyzed monovalent oxidation of ascorbate to ascorbate free radicals (Asc^?). The O₂^?? generation and Asc^? formation were detected by O₂^??-specific chemiluminescence of the Cypridina lucigenin analog and electron spin resonance spectroscopy, respectively. PEA-dependent O₂^?? production and monovalent oxidation of ascorbate in the Hb solution occurred without addition of H₂O₂, but a trace of H₂O₂ added to the system greatly increased the production of both O₂^?? and Asc^?. Addition of GSH completely inhibited the PEA-dependent production of O₂^?? and Asc^? in Hb solution. We propose that the O₂^?? generation and Asc^? formation in the Hb solution are due to the pseudoperoxidase activity-dependent oxidation of PEA and resultant ROS may damage tissues rich in monoamines, if the Hb-based blood substitutes were circulated without addition of ROS scavengers such as thiols.     

31P NUCLEAR MAGNETIC RESONANCE STUDY OF INTRACELLULAR PHOSPHATE POOLS AND POLYPHOSPHATE METABOLISM IN HETEROSIGMA AKASHIWO (HADA) HADA (RAPHIDOPHYCEAE)1

The effects of starvation and subsequent addition of phosphate-containing medium on the phosphate metabolic intermediates were studied by ³¹P-NMR spectroscope of perchloric acid extracts and intact cells of Heterosigma akashiwo (Hada) Hada. When orthophosphate in the medium was completely depleted the medium was enriched with orthophosphate (4.5 μM). In the phosphate starved condition, the P cell quota was 76 fmol·cell⁻¹ and the major components of phosphate intermediates were phosphodiester, sugar phosphate and orthophosphate (P_i). After addition of P_i, rapid uptake of P_i was observed and the P cell quota increased to 108 fmol·cell⁻¹ in 2 h, 134 fmol·cell⁻¹ in 5 h and 222 fmol·cell⁻¹ in 1 day after addition of phosphate. The ³¹P-NMR spectrum indicated that a major portion of P was stored as polyphosphate, in which the average chain length of polyphosphate increased from 10 to 20 phosphate residues in one day after addition of P_i.     

DESCRIPTION OF VIRIDILOBUS MARINUS (GEN. ET SP. NOV.), A NEW RAPHIDOPHYTE FROM DELAWARE'S INLAND BAYS

Delaware's Inland Bays (DIB), USA, are subject to blooms of potentially harmful raphidophytes, including Heterosigma akashiwo. In 2004, a dense bloom was observed in a low salinity tributary of the DIB. Light microscopy initially suggested that the species was H. akashiwo; however, the cells were smaller than anticipated. 18S rDNA sequences of isolated cultures differed substantially from all raphidophyte sequences in GenBank. Phylogenetic analysis placed it approximately equidistant from Chattonella and Heterosigma with only ~96% sequence homology with either group. Here, we describe this marine raphidophyte as a novel genus and species, Viridilobus marinus (gen. et sp. nov.). We also compared this species with H. akashiwo, because both species are superficially similar with respect to morphology and their ecological niches overlap. V. marinus cells are ovoid to spherical (11.4 × 9.4 μm), and the average number of chloroplasts (4 per cell) is lower than in H. akashiwo (15 per cell). Pigment analysis of V. marinus revealed the presence of fucoxanthin, violaxanthin, and zeaxanthin, which are characteristic of marine raphidophytes within the family Chattonellaceae of the Raphidophyceae. TEM and confocal microscopy, however, revealed diagnostic microscopic and ultrastructural characteristics that distinguish it from other raphidophytes. Chloroplasts were in close association with the nucleus and thylakoids were arranged either parallel or perpendicular to the cell surface. Putative mucocysts were identified, but trichocysts were not observed. These features, along with DNA sequence data, distinguish this species from all other raphidophyte genera within the family Chattonellaceae of the Raphidophyceae.     

IDENTIFICATION OF THE TOXIC DINOFLAGELLATES ALEXANDRIUM CATENELLA AND A. TAMARENSE (DINOPHYCEAE) USING DNA PROBES AND WHOLE-CELL HYBRIDIZATION1

Fluorescent DNA probes (cCAT-F1 and cTAM-Fl) complementary to the 3′ end of ribosomal RNA (rRNA) internal transcribed spacer 1 sequences (ITS 1: positions 154–176) of toxic species of Alexandrium catenella (Whedon and Kofoid) Taylor and A. tamarense (Lebour) Taylor were applied to various cultures of the genus Alexandrium and several other phytoplankters using whole-cell fluorescent in situ hybridization. cCAT-F1 and cTAM-F1 reacted with targeted strains of A. catenella (catenella type) and A. tamarense (tamarense type), respectively, and did not react with isolates of A. affine (Inoue et Fukuyo) Balech, A. fraterculus (Balech) Balech, A. insuetum Balech, A. lusitanicum Balech, A. pseudogonyaulux (Biecheler)Horiguchi ex Yuki et Fukuyo comb. nov., nor isolates of Prorocentrum micans Ehrenberg, Amphidinium carterae Hulburt, Heterocapsa triquetra (Ehrenberg) Stein, Gymnodinium mikimotoi Miyake et Kominami ex Oda, Skeletonema costatum (Greville) Cleve, Heterosigma akashiwo (Hada) Hada, and Chattonella antiqua (Hada) Ono. DNase I and RNase A treatment showed that probes hybridized to ribosomal DNA, not rRNA. Probes were localized at the bottom of the U-shaped nucleus, a region that corresponds to the nucleolus. The probes are highly specific for particular strains of A. catenella and A. tamarense and are applicable for identifying these species collected from cultured and possibly natural populations.