Dinoflagellate Scrippsiella Trochoidea Research Articles

Cloning and comparative studies of proliferating cell nuclear antigen (PCNA) genes for nine dinoflagellates

Proliferating cell nuclear antigen (PCNA), a co-factor of DNA polymerases δ and e, is associated with active cell proliferation, particularly with the S phase of the eukaryotic cell cycle, and the expression of this gene has been therefore proposed as a molecular marker for estimating phytoplankton growth rate and even population dynamics. In this study, the full-length cDNA sequences of PCNA for nine dinoflagellate species (ten strains) were obtained, analyzed, and further characterized with the expression in response to alterations of growth stage and life cycle. All the ten genes have a 777 or 780 bp ORF and encode a protein of 258 or 259 amino acids, similar to PCNAs from vertebrates and plants. The deduced amino acid sequences include conservative motif characteristic to the PCNA gene family, implying conserved functions of PCNA among organisms of different taxa. Alignment analysis in combination with other sequences available in NCBI database exhibited high amino acid similarities (79.2–96.9%) among PCNAs from 17 dinoflagellate species. Phylogenetic tree derived from 37 PCNA sequences of dinoflagellates was consistent with the taxonomic affiliation of these taxa. Then, we used the cosmopolitan, toxic, and resting cyst-producing dinoflagellate Scrippsiella trochoidea as a representative of HAB-forming dinoflagellates to investigate PCNA (StPCNA) expression at different stages of growth and life cycle and the results of real-time quantitative PCR revealed clearly a stage-dependent pattern in the transcription of StPCNA. The transcripts dramatically reduced from an exponential to a stationary growth stage and then further significantly decreased from a stationary stage to dormant stage (resting cysts), a pattern in accordance with that previously reported from the dinoflagellates Alexandrium catenella and Prorocentrum donghaiense. In terms of its minimal implication, this work enhances the previous perception about the function and possible application of PCNA gene, and more importantly, our results indicated that PCNA is not silent during the dormant stage of dinoflagellates.

Molecular cloning of heat shock protein 60 (Hsp60) and 10 (Hsp10) genes from the cosmopolitan and harmful dinoflagellate Scrippsiella trochoidea and their differential transcriptions responding to temperature stress and alteration of life cycle

Heat shock protein 60 (Hsp60) and Hsp10 are two chaperones important to both stress responses and cellular metabolisms in most organisms. In this study, the cosmopolitan Scrippsiella trochoidea was used as a model of HAB-forming dinoflagellates to explore the possible functional roles of Hsp60 and Hsp10 in the adaptation of dinoflagellates to temperature stress and life cycle transition. The full-length cDNAs of a Hsp60 and a Hsp10 gene from S. trochoidea (StHsp60 and StHsp10) were obtained via rapid amplification of cDNA ends (RACE) and their deduced amino acid sequences both included family-characteristic conservative structures and motifs, indicating a conserved function for both among different taxa. Real-time qPCR revealed that StHsp60 and StHsp10 exhibited highly similar mRNA accumulation patterns in response to temperature stresses. Their mRNA amounts, compared to that at 20 °C (control), were rapidly up-regulated upon exposure to both lower (15 °C, 10 °C, 5 °C) and higher (25 °C, 30 °C) temperatures and showed a clear time-dependent manner, suggesting a possible involvement of StHsp60 and StHsp10 in the urgent adaptation of S. trochoidea to drastic temperature stress. Furthermore, significantly elevated mRNA levels of both genes were detected in resting cysts (newly formed and that maintained in dormancy for different durations) relative to that in vegetative cells, suggesting that higher levels of StHsp60 and StHsp10 are demanded by S. trochoidea resting cysts. The results of this work, as the first investigation to characterize Hsp60 and Hsp10 genes from dinoflagellates, enrich the knowledge about Hsps and lay an important foundation for further probing their functions in dinoflagellate resting cysts.

Cloning and Partial Characterization of a Cold Shock Domain-Containing Protein Gene from the Dinoflagellate Scrippsiella trochoidea.

CSPs, cold shock domain (CSD) containing proteins, are demonstrated to be involved in low temperature responses and various cellular processes under normal growth conditions. Here, we used the cosmopolitan, toxic, and resting cyst-producing dinoflagellate Scrippsiella trochoidea as a representative harmful algal bloom-forming dinoflagellate to investigate the expression patterns of CSP in vegetative cells in response to temperature shocks and in resting cysts, with an objective to probe the possible function of CSP in dinoflagellates. The full-length cDNA of a CSP gene from S.trochoidea (StCSP) was obtained which has a solely N-terminal CSD with conserved nucleic acids binding motifs. The qPCR results together indicated StCSP expression was not modulated by temperature at the transcriptional level and implied this gene may not be associated with temperature stress responses in S.trochoidea as the gene's name implies. However, we observed significantly higher StCSP transcripts in resting cysts (newly formed and maintained in dormancy for different periods of time) than that observed in vegetative cells (at exponential and stationary stages), indicating StCSP is actively expressed during dormancy of S.trochoidea. Taking together our recent transcriptomic work on S.trochoidea into consideration, we postulate that StCSP may play roles during encystment and cyst dormancy of the species.

Metabolomic Fingerprints of Individual Algal Cells Using the Single-Probe Mass Spectrometry Technique.

Traditional approaches for the assessment of physiological responses of microbes in the environment rely on bulk filtration techniques that obscure differences among populations as well as among individual cells. Here, were report on the development on a novel micro-scale sampling device, referred to as the “Single-probe,” which allows direct extraction of metabolites from living, individual phytoplankton cells for mass spectrometry (MS) analysis. The Single-probe is composed of dual-bore quartz tubing which is pulled using a laser pipette puller and fused to a silica capillary and a nano-ESI. For this study, we applied Single-probe MS technology to the marine dinoflagellate Scrippsiella trochoidea, assaying cells grown under different illumination levels and under nitrogen (N) limiting conditions as a proof of concept for the technology. In both experiments, significant differences in the cellular metabolome of individual cells could readily be identified, though the vast majority of detected metabolites could not be assigned to KEGG pathways. Using the same approach, significant changes in cellular lipid complements were observed, with individual lipids being both up- and down-regulated under light vs. dark conditions. Conversely, lipid content increased across the board under N limitation, consistent with an adjustment of Redfield stoichiometry to reflect higher C:N and C:P ratios. Overall, these data suggest that the Single-probe MS technique has the potential to allow for near in situ metabolomic analysis of individual phytoplankton cells, opening the door to targeted analyses that minimize cell manipulation and sampling artifacts, while preserving metabolic variability at the cellular level.

Profile of Citrobacter freundii ST2, a Multi-acyl-homoserine Lactone Producer Associated with Marine Dinoflagellates.

Marine algae provide a unique niche termed the phycosphere for microorganism inhabitation. The phycosphere environment is an important niche for mutualistic and competitive interactions between algae and bacteria. Quorum sensing (QS) serves as a gene regulatory system in the microbial biosphere that allows bacteria to sense the population density with signaling molecules, such as acyl-homoserine lactone (AHL), and adapt their physiological activities to their surroundings. Understanding the QS system is important to elucidate the interactions between algal-associated microbial communities in the phycosphere condition. In this study, we isolated an epidermal bacterium (ST2) from the marine dinoflagellate Scrippsiella trochoidea and evaluated its AHL production profile. Strain ST2 was classified as a member of the genus Citrobacter closely related to Citrobacter freundii by 16S rRNA gene sequence analysis. Thin-layer chromatography revealed that C. freundii ST2 secreted three active AHL compounds into the culture supernatant. Specific compounds, such as N-butyryl-L-homoserine lactone (C4-AHL), N-octanoyl-DL-homoserine lactone (C8-AHL), and N-decanoyl-DL-homoserine lactone (C10-AHL), were identified by high-resolution tandem mass spectrometry. Carbon metabolic profiling with Biolog EcoPlate™ indicated that C. freundii ST2 was widely used as a carbon source and preferred carbohydrates, amino acids, and carboxylic acids as carbon substrates. Our results demonstrated that C. freundii ST2 is a multi-AHL producer that participates in the phycosphere carbon cycle.

Genome sequence of Enterobacter sp. ST3, a quorum sensing bacterium associated with marine dinoflagellate.

Phycosphere environment is a typical marine niche, harbor diverse populations of microorganisms, which are thought to play a critical role in algae host and influence mutualistic and competitive interactions. Understanding quorum sensing-based acyl-homoserine lactone (AHL) language may shed light on the interaction between algal-associated microbial communities in the native environment. In this work, we isolated an epidermal bacterium (was tentatively named Enterobacter sp. ST3, and deposited in SOA China, the number is MCCC1K02277-ST3) from the marine dinoflagellate Scrippsiella trochoidea, and found it has the ability to produce short-chain AHL signal. In order to better understand its communication information at molecular level, the genomic map was investigated. The genome size was determined to be 4.81 Mb with a G + C content of 55.59%, comprising 6 scaffolds of 75 contigs containing 4647 protein-coding genes. The functional proteins were predicted, and 3534 proteins were assigned to COG functional categories. An AHL-relating gene, LuxR, was found in upstream position at contig 1. This genome data may provide clues to increase understanding of the chemical characterization and ecological behavior of strain ST3 in the phycosphere microenvironment.

A comparative study of maximal quantum yield of photosystem II to determine nitrogen and phosphorus limitation on two marine algae

We studied the effects of nitrogen and phosphorus supply on Fv/Fm (maximal quantum yield of photosystem II) in the diatom Chaetoceros debilis and dinoflagellate Scrippsiella trochoidea in nitrogen (N) and phosphorus (P) depleted cultures to determine whether this parameter could be used to monitor N or P limitation. In the nutrient depleted experiments, no obvious decrease of cell density and chlorophyll concentration was observed except in N-depleted incubation of S. trochoidea. For C. debilis, Fv/Fm decreased quickly in periods of N- and P-depletion and re-supply of N and P induced a quick recovery of Fv/Fm. However, in S. trochoidea culture, Fv/Fm remained unchanged in N- and P-depleted conditions and addition of sufficient N and P to N- and P-depleted cultures did not affect Fv/Fm. Therefore, Fv/Fm is insensitive to N and/or P limitation in growth of S. trochoidea. The results suggested that Fv/Fm was not a robust diagnostic for nutrient limitation in dinoflagellates. The differences in the sensitivity of Fv/Fm to nutrient limitation may result from different nutrient storage abilities among algal species.

Metabolism of DMSP, DMS and DMSO by the cultivable bacterial community associated with the DMSP-producing dinoflagellate Scrippsiella trochoidea

Bacterial species associated with the dimethylsulfoniopropionate (DMSP)-producing phytoplankton Scrippsiella trochoidea were cultured and identified, with the aim of establishing their ability to metabolise DMSP, dimethylsulfide (DMS) and dimethylsulfoxide (DMSO). Results demonstrate that of the cultivable bacteria only α-Proteobacteria were capable of producing DMS from DMSP. The concentration of DMSP was shown to affect the amount of DMS produced. Lower DMSP concentrations (1.5 μmol dm−3) were completely assimilated, whereas higher concentrations (10 μmol dm−3) resulted in increasing amounts of DMS being produced. By contrast to the restricted set of bacteria that metabolised DMSP, ~ 70% of the bacterial isolates were able to ‘consume’ DMS. However, 98-100% of the DMS removed was accounted for as DMSO. Notably, a number of these bacteria would only oxidise DMS in the presence of glucose, including members of the γ-Proteobacteria and Bacteroidetes. The observations from this study, coupled with published field data, identify DMS oxidation to DMSO as a major transformation pathway for DMS, and we speculate that the fate of DMS and DMSP in the field are tightly coupled to the available carbon produced by phytoplankton.

Lethal effects of Northwest Atlantic Ocean isolates of the dinoflagellate, Scrippsiella trochoidea, on Eastern oyster (Crassostrea virginica) and Northern quahog (Mercenaria mercenaria) larvae

The thecate dinoflagellate Scrippsiella trochoidea is a cosmopolitan, bloom-forming alga that has been generally considered non-toxic. Here, we report that environmentally relevant cell densities (104 cells mL−1) of Scrippsiella trochoidea strains isolated from the Northwest Atlantic Ocean caused 100% mortality in Eastern oyster (Crassostrea virginica) larvae during 3-day exposures while parallel control larvae exhibited 100% survival. S. trochoidea also exhibited lethal effects on Northern quahog (Mercenaria mercenaria) larvae (70% mortality during 3-day exposure) but were non-toxic to juvenile fish (Cyprinodon variegates). The cultures of S. trochoidea were more lethal to Northern quahog larvae than ten other species of harmful algae, including the highly toxic species Cochlodinium polykrikoides. Scrippsiella trochoidea cultures within later stages of growth were more toxic than exponential growth stages to bivalve larvae, and the toxicity was dose dependent. Furthermore, toxicity was maintained in the cultures that were sonicated, boiled, and frozen as well as in resuspended residues of the culture but was significantly lower in cell-free culture media. Collectively, these results suggest that S. trochoidea causes mortality in bivalve larvae through a physicochemical rather than strictly chemical mechanism, such as clogging of larval feeding apparatuses by materials produced by S. trochoidea (e.g., lipids, extracellular polysaccharides, and/or cell debris) which accumulate as cells in culture or blooms age. This is the first report of the lethal effects of Scrippsiella trochoidea on shellfish larvae.

Interplay Between the Parasite Amoebophrya sp. (Alveolata) and the Cyst Formation of the Red Tide Dinoflagellate Scrippsiella trochoidea

Syndiniales (Alveolata) are marine parasites of a wide range of hosts, from unicellular organisms to Metazoa. Many Syndiniales obligatorily kill their hosts to accomplish their life cycle. This is the case for Amoebophrya spp. infecting dinoflagellates. However, several dinoflagellate species known to be infected by these parasites produce diploid resting cysts as part of their life history. These resting cysts may survive several seasons in the sediment before germinating. How these parasites survive during the dormancy of their host remained an open question. We successfully established infections by Amoebophrya sp. in the red tide dinoflagellate Scrippsiella trochoidea. This host strain was homothallic and able to continuously produce typical calcified cysts covered by calcareous spines. Presence of the parasite significantly speeded up the host cyst production, and cysts produced were the only cells to resist infections. However, some of them were clearly infected, probably earlier in their formation. After 10 months, cysts produced in presence of the parasite were able to germinate and new infective cycles of the parasite were rapidly observed. Thus, a very novel relationship for protists is demonstrated, one in which parasite and host simultaneously enter dormancy, emerging months later to propagate both species.

Transformation of iodate to iodide in marine phytoplankton driven by cell senescence

AB Aquatic Biology Contact the journal Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsTheme Sections AB 11:1-15 (2010) - DOI: https://doi.org/10.3354/ab00284 FEATURE ARTICLE Transformation of iodate to iodide in marine phytoplankton driven by cell senescence K. Bluhm1,*, P. Croot1, K. Wuttig1, K. Lochte2 1IFM-GEOMAR, Leibniz Institute for Marine Sciences, Marine Biogeochemistry, Westshore Building, Duesternbrooker Weg 20, 24105 Kiel, Germany 2Alfred-Wegener-Institute for Polar- and Marine Research, Am Handelshafen 12, 27570 Bremerhaven, Germany *Email: kbluhm@ifm-geomar.de ABSTRACT: Previous studies have suggested that phytoplankton play an important role in the biogeochemical cycling of iodine, due to the appearance of iodide in the euphotic zone. Changes in the speciation of iodine over the course of the growth cycle were examined in culture media for a variety of phytoplankton taxa (diatoms, dinoflagellates and prymnesiophytes). All species tested showed the apparent ability to reduce iodate to iodide, though production rates varied considerably between species (0.01 to 0.26 nmol l–1 µg–1 chl a d–1), with Eucampia antarctica the least and Pseudo-nitzschia turgiduloides the most efficient iodide producers. Production was found to be species specific and was not related to biomass (indicated by e.g. cell size, cell volume, or chl a content). In all species, except for the mixotrophic dinoflagellate Scrippsiella trochoidea, iodide production commenced in the stationary growth phase and peaked in the senescent phase of the algae, indicating that iodide production is connected to cell senescence. This suggests that iodate reduction results from increased cell permeability, which we hypothesize is due to subsequent reactions of iodate with reduced sulphur species exuded from the cell. A shift from senescence back to the exponential growth phase resulted in a decline in iodide and indicated that phytoplankton-mediated oxidation of iodide to iodate was likely to be occurring. Iodide production could not be observed in healthy cells kept in the dark for short periods. Bacterial processes appeared to play only a minor role in the reduction of iodate to iodide. KEY WORDS: Iodine speciation · Iodide · Iodate · Antarctic diatoms · Nitrate reductase · Glutathione · Sulphur species · Cell senescence Full text in pdf format Information about this Feature Article NextCite this article as: Bluhm K, Croot P, Wuttig K, Lochte K (2010) Transformation of iodate to iodide in marine phytoplankton driven by cell senescence. Aquat Biol 11:1-15. https://doi.org/10.3354/ab00284 Export citation RSS - Facebook - Tweet - linkedIn Cited by Published in AB Vol. 11, No. 1. Online publication date: October 28, 2010 Print ISSN: 1864-7782; Online ISSN: 1864-7790 Copyright © 2010 Inter-Research.

The phytoplankton of Guanabara Bay, Brazil: I. historical account of its biodiversity

This is a historical account of the biodiversity of phytoplankton in Guanabara Bay, Brazil. It is based on 57 publications that refer to sampling carried out between 1913 and 2004. The publications included are those with direct microscopic identification. Although 80% of the studies focus on ecological issues that tend to mention only the most abundant species, 24 publications provide comprehensive check-lists at the species level, especially of taxa > 20 μm. The inventory of species includes, to date, 308 taxa among 199 diatoms, 90 dinoflagellates, 9 cyanobacteria, 5 euglenophyceans, 1 chlorophycean, 1 prasinophycean, 1 silicoflagellate, and 2 ebriids. The most conspicuous species were the dinoflagellate Scrippsiella trochoidea and diatoms from the Skeletonema costatum complex. The first was the theme of the very first publication in the area (Faria 1914) that reported on its bloom associated with the mass mortality of fish due to oxygen depletion; it is still often found in high abundances (10(6) cell.L-1) in more protected areas. The second was long considered in the literature as a cosmopolitan and opportunistic species, until the recent discovery of cryptic species within the genus; taxonomic re-evaluation of local populations is, therefore, needed. Besides these two species, only other 25 species stood out in terms of frequency of occurrence and widespread distribution in the Bay, some known to be implicated in harmful blooms elsewhere. The biodiversity of dinoflagellates, especially within the Gymnodiniales, and that of other unidentified flagellates (Haptophyceae, Cryptophyceae, Prasinophyceae, Raphidophyceae) is largely underestimated because of the use of fixatives that distort/destroy diagnostic characters. From the initial inventory of 124 taxa published in 1917 and the subsequent additions in species numbers, one can have a false perception that the phytoplankton biodiversity has increased throughout the years, despite the overall increase in eutrophication observed in Guanabara Bay. The reason for this may be twofold: 1) it is an artifact caused by our progressively improving technical capability to detect and identify species and 2) the possible effects of eutrophication could be better perceived when the community structure is evaluated, that is, when space-time variations in the abundances of the populations (rather than just number of species) are also taken into account.

Characteristics of phytoplankton community structure during and after a bloom of the dinoflagellate Scrippsiella trochoidea by HPLC pigment analysis

A bloom of the dinoflagellate Scrippsiella trochoidea was detected for the first time in inner Tolo Harbor, Hong Kong in 2 000. Water samples were collected at eight stations along a transect passing through a red tide patch for microscopic analysis of phytoplankton composition and high-performance liquid chromatography (HPLC) analysis of phytoplankton pigments. During the bloom, the density of dinoflagellates was 1.1×106 cells L−1 within the patch and 8.6×105 cells L−1 outside the patch where the phytoplankton community was dominated by diatoms. After the bloom the S. trochoidea began to decrease in density and was replaced by diatoms as the dominating bloom-causing organisms at all stations, and the density of dinoflagellates at most stations was less than 1.0 × 106 cells L−1. The status of S. trochoidea as the causative species of the bloom was indicated by the presence of peridinin, the marker pigment for dinoflagellates. The shift from dinoflagellates to diatoms was marked by the decline of peridinin and the prevalence of fucoxanthin. Phytoplankton pigment markers also revealed the presence of other minor phytoplankton assemblages such as cryptomonads and blue-green algal.

Inactivation of dinoflagellate Scrippsiella trochoidea in synthetic ballast water by reactive species generated from dielectric barrier discharges

The inactivation of dinoflagellate Scrippsiella trochoidea in synthetic ballast water by a dielectric barrier discharge (DBD) system was investigated. The radical, ozone and hydrogen peroxide generated from the DBD system were measured. Before and after the treatment, the viability of dinoflagellate S. trochoidea was evaluated by analyzing chlorophyll a, protein and saccharide content and morphology of the cells, as well as the pH of the cell culture media. The results show that radical was the major reactive species when humid air was used. The inactivation of S. trochoidea was found to be dependent on the applied voltage and the gas flow rate, and was completed within 4 min at a gas flow rate of 7 L min−1 and an applied voltage of 20 kV. The change of chlorophyll a, protein and saccharide concentrations of S. trochoidea and the morphology of the cells indicates that the reactive species generated from the DBD system can break up the cells via oxidation.

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

Red tides (high biomass phytoplankton blooms) have frequently occurred in Hong Kong waters, but most red tides occurred in waters which are not very eutrophic. For example, Port Shelter, a semi-enclosed bay in the northeast of Hong Kong, is one of hot spots for red tides. Concentrations of ambient inorganic nutrients (e.g. N, P), are not high enough to form the high biomass of chlorophyll a (chl a) in a red tide when chl a is converted to its particulate organic nutrient (N) (which should equal the inorganic nutrient, N). When a red tide of the dinoflagellate Scrippsiella trochoidea occurred in the bay, we found that the red tide patch along the shore had a high cell density of 15,000 cells ml −1, and high chl a (56 μg l −1), and pH reached 8.6 at the surface (8.2 at the bottom), indicating active photosynthesis in situ. Ambient inorganic nutrients (NO 3, PO 4, SiO 4, and NH 4) were all low in the waters and deep waters surrounding the red tide patch, suggesting that the nutrients were not high enough to support the high chl a >50 μg l −1 in the red tide. Nutrient addition experiments showed that the addition of all of the inorganic nutrients to a non-red-tide water sample containing low concentrations of Scrippsiella trochoidea did not produce cell density of Scrippsiella trochoidea as high as in the red tide patch, suggesting that nutrients were not an initializing factor for this red tide. During the incubation of the red tide water sample without any nutrient addition, the phytoplankton biomass decreased gradually over 9 days. However, with a N addition, the phytoplankton biomass increased steadily until day 7, which suggested that nitrogen addition was able to sustain the high biomass of the red tide for a week with and without nutrients. In contrast, the red tide in the bay disappeared on the sampling day when the wind direction changed. These results indicated that initiation, maintenance and disappearance of the dinoflagellate Scrippsiella trochoidea red tide in the bay were not directly driven by changes in nutrients. Therefore, how nutrients are linked to the formation of red tides in coastal waters need to be further examined, particularly in relation to dissolved organic nutrients.

Characterization of azaspiracids in plankton size-fractions and isolation of an azaspiracid-producing dinoflagellate from the North Sea

Azaspiracids (AZAs) are a group of lipophilic polyether toxins implicated in incidents of shellfish poisoning in humans, particularly in northern Europe. In an attempt to establish the biogeographical distribution of AZA toxins, their association with plankton size-fractions, and to confirm the identity of the causative species responsible for human poisoning, a month-long oceanographic study was undertaken in coastal North Sea waters. The occurrence and abundance of AZA analogues was measured by on board triple quadrupole mass spectrometry coupled to liquid chromatography (LC-MS/MS). In size-fractionated plankton samples collected by net tows (20 μm mesh-size), by pumping from discrete depths and from Niskin entrapment bottle casts to fixed depths, AZA-1 was consistently the major azaspiracid component. In eastern Scottish coastal waters, the highest amounts of AZA-1 in net tow samples were in the 50–200 μm fractions, with lesser amounts detected in the >200 μm and 50–20 μm fractions. At these stations, the 50–200 μm fractions were rich in the ciliate Favella ehrenbergii. Cells of F. ehrenbergii isolated by microcapillary indeed contained AZA-1, but isolated cells grown and fed the non-toxic dinoflagellate Scrippsiella trochoidea for one week failed to contain any detectable AZA-1—evidence that F. ehrenbergii is merely a vector for AZA. Detailed analysis of plankton from Niskin bottle samples from around the North Sea typically showed highest amounts of AZA in the 3–20 μm fraction. From this fraction, a large number of crude cultures were established and subsequently screened for the presence of AZAs. A small photosynthetic thecate dinoflagellate, provisionally designated as strain 3D9, was isolated by microcapillary and brought into pure culture. This dinoflagellate strain was found to produce AZA-1, AZA-2 and an isomer of AZA-2. Sequence comparisons by molecular genetic techniques also indicated that this genotype was present in field samples rich in AZA. This discovery of a novel causative dinoflagellate for AZA toxicity essentially explains the lack of correlation of AZA with the abundance and distribution of the previously postulated culprit species Protoperidinium crassipes. We instead propose that such large phagotrophic dinoflagellates can act as an AZA vector following grazing upon a proximal source, such as the dinoflagellate 3D9 strain.

Harmful effects of the toxic dinoflagellate Alexandrium tamarense on the tintinnids Favella taraikaensis and Eutintinnus sp.

The effects of the exposure of two tintinnids (Favella taraikaensis and Eutintinnus sp.) to the toxic dinoflagellate Alexandrium tamarense were studied, using the non-toxic dinoflagellate Scrippsiella trochoidea as a control. The tintinnid F. taraikaensis showed backward swimming and cellular lysis after a few minutes in the presence of the toxic dinoflagellate A. tamarense in high concentration (280–700 cells ml-1). At low concentrations of A. tamarense (30–120 cells ml-1) no swimming alterations were observed, but cellular lysis occurred after 48 hours. No changes in the swimming patterns were observed when Eutintinnus sp. was exposed to either toxic dinoflagellates or cell-free filtrate, or when F. taraikaensis was exposed to cell-free filtrate. However, both tintinnids suffered cellular lysis after 48 hours of exposure. Favella taraikaensis and Eutintinnus sp. remained healthy after preying on the non-toxic S. trochoidea for 48 hours. In selectivity experiments, F. taraikaensis avoided cells of the toxic dinoflagellates during the dark phase. Our results suggest that tintinnids could play a regulatory role when the toxic blooms are initiating and the toxic cell concentrations remain still low in the northern Patagonian gulfs of Argentina. Since tintinnids are affected by substances released by A. tamarense, predation on dinoflagellates would not occur during the peak of the bloom.

Algae viability within copepod faecal pellets: evidence from microscopic examinations

The viability of different phytoplankton species within copepod faecal pellets was tested. Monocultures of the dinoflagellate Scrippsiella trochoidea and the diatoms Fragilariopsis ker- guelensis, Proboscia alata and Chaetoceros dichaeta were fed to Calanus helgolandicus, Temora longicornis and Centropages typicus. Microscopic observations of faecal pellet content, chlorophyll auto-fluorescence and the fluorescence of cells stained with the viability stain fluorescein diacetate (FDA) revealed that some S. trochoidea cells survived the gut passage of T. longicornis and C. typi- cus. Viable cells of F. kerguelensis were observed in faecal pellets of all copepod species investi- gated. The 2 diatom species P. alata and C. dichaeta were reduced to small fragments within all faecal pellets, and hence did not survive grazing by any of the 3 copepod species.

On the allelochemical potency of the marine dinoflagellate Alexandrium ostenfeldii against heterotrophic and autotrophic protists

Three strains of the marine dinoflagellate Alexandrium ostenfeldii of different geographic origin were tested for their short-term deleterious effects on a diversity of marine protists. All A. ostenfeldii strains were capable of eliciting an apparent allelochemical response, but the various protistan target species were differentially affected. Protists that were negatively affected by exposure to cells of A. ostenfeldii and associated extracellular metabolites comprised both autotrophs (Rhodomonas sp., Dunaliella salina, Thalassiosira weissflogii) and heterotrophs (Oxyrrhis marina, Amphidinium crassum, Rimostrombidium caudatum). Observed effects included immobilisation (e.g. of O. marina), morphological changes (e.g. in D. salina) and/or aberrant behaviour (e.g. of R. caudatum), mainly as preliminary stages of cell lysis. Immobilization and lytic effects against O. marina were strongly dependent on A. ostenfeldii cell concentrations. Effects also differed substantially among strains and different batch cultures of the same strain. Values of EC50, defined as the A. ostenfeldii cell concentration causing lysis of 50% of O. marina cells, ranged from 0.3 to 1.9 � 10 3 mL 21 , depending on the A. ostenfeldii strain. The autotrophic dinoflagellate Scrippsiella trochoidea reacted to exposure to A. ostenfeldii cells by formation of temporary (ecdysal) cysts, whereas, in contrast, the flagellates Emiliania huxleyi and Prymnesium parvum and the ciliate Strombidium sp. were relatively refractory or even unaffected. As long as cells did not lyse, the fluorescence yield of target autotrophs, estimated by pulse-amplitude modulation fluorometry, did not significantly change during the first 3 h of incubation, suggesting that allelochemicals produced by A. ostenfeldii caused no shortterm negative effects on the photosynthetic apparatus. Overall, the allelochemical responses of target species showed no obvious relationship to cell quota or extracellular concentrations of either toxic macrocyclic imines (spirolides) or tetrahydropurine neurotoxins (saxitoxin and analogues) produced by various strains of A. ostenfeldii. Instead, the potency of A. ostenfeldii, eliciting immobilization and lytic species-specific responses in potential predators and competitors, is consistent with the existence of an allelochemical mechanism unrelated to the bioactivity of known phycotoxins of the genus Alexandrium.

Microalgal lipid markers for paleoclimatic research

Primary producers, mainly represented in the marine environment by microalgae, synthesize a wide variety of lipid constituents, some of which are recognized as ‘biomarkers’ that can be used to identify sources of organic matter. Furthermore, as phytoplankton rapidly reflect climate variations, the identification of organic matter in sediments may help to understand present or ancient specific environmental conditions. To highlight differences in some lipid constituents (hydrocarbons, sterols and fatty acids) of different taxa of microalgae, we determined these compounds in the diatom Cylindrotheca closterium and dinoflagellate Scrippsiella trochoidea isolated from the Adriatic Sea and grown in batch cultures. Total lipids were extracted in chloroform-methanol and, after separation, analysed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). The only hydrocarbons present in these species were cis-3,6,9,12,15,18-heneicosahexaene (HEH) and squalene. Analysis of sterols in C. closterium revealed that only 4-desmethylsterols were present. Conversely, in S. trochoidea, 2 different groups of sterols (4-desmethylsterols and 4a-methylsterols) were identified. Besides the fatty acid 16:0, which predominated in both species, high percentages of 16:1n-7 and 20:5n-3 were observed in C. closterium, whereas 18:4n-3 and 22:6n-3 were detected in S. trochoidea. Differences in lipid patterns were observed between C. closterium and S. trochoidea. These differences might help understand the Adriatic Sea Basin’s paleoclimatic history.