Rhodopsins build up the birefringent bodies of the dinoflagellate Oxyrrhis marina

The ultrastructure of the birefringent bodies of the dinoflagellate Oxyrrhis marina was investigated by transmission electron microscopy. Ultrathin sectioning revealed that the bodies consist of highly ordered and densely packed lamellae, which show a regular striation along their longitudinal axis. A lattice distance of 6.1 nm was measured for the densely packed lamellae by FFT (Fast Fourier Transformation) analysis. In addition, a rather faint and oblique running striation was registered. Lamellae sectioned rather oblique or almost close to the surface show a honeycombed structure with a periodicity of 7.2–7.8 nm. Freeze-fracture transmission electron microscopy revealed that the lamellae are composed of highly ordered, crystalline arrays of particles. Here, FFT analysis resulted in lattice distances of 7.0–7.6 nm. Freeze-fracture transmission electron microscopy further revealed that the bodies remained intact after cell rupture followed by ascending flotation of the membrane fractions on discontinuous sucrose gradients. The birefringent bodies most likely are formed by evaginations of membranes, which separate the cytoplasm from the food vacuoles. Distinct, slightly reddish-colored areas, which resembled the birefringent bodies with respect to size and morphology, were registered by bright field light microscopy within Oxyrrhis marina cells. An absorbance maximum at 540 nm was registered for these areas, indicating that they are composed of rhodopsins. This was finally proven by immuno-transmission electron microscopy, as antisera directed against the C-terminal amino acid sequences of the rhodopsins AEA49880 and ADY17806 intensely immunolabeled the birefringent bodies of Oxyrrhis marina.

     

IMMUNOLOCALIZATION OF CENTRIN IN OXYRRHIS MARINA (DINOPHYCEAE)1

A new polyclonal antibody was raised against centrin isolated from the flagellate green alga Spermatozopsis similis (Chlorophyta; anti-SSC). It stains by immunofluorescence and immunoelectron microscopy well-known reference systems for centrin like the nucleus–basal body connectors in Chlamydomonas reinhardtii (Chlorophyta) and the system II fibers (rhizoplasts) of Scherffelia dubia (Chlorophyta). In addition, it recognizes in immunoblots a single 20-kDa protein in isolated cytoskeletons of Spermatozopsis similis and Tetraselmis striata (Chlorophyta) as well as purified centrin isolated from Tetraselmis striata. Using this antibody, centrin was localized in whole cells and isolated cytoskeletons of Oxyrrhis marina Dujardin (Dinophyceae) by immunofluorescence and immunogold electron microscopy. In the flagellar apparatus of O. marina, five different structures were antigenic. Four short fibers (connectives 1–4) link the basal bodies to the four major fibrous flagellar roots, which do not cross-react with anti-centrin. The most prominent of the labeled structures (connective 5), a crescent-shaped fiber, extends from the flagellar canal of the transverse flagellum along the base of the tentacle to the flagellar canal of the longitudinal flagellum, interconnecting the distal parts of the microtubular roots/bands in the basal apparatus. For most of its length, it underlies and is connected to a transversely oriented subamphiesmal microtubular band. In immunoblot analyses, anti-SSC recognizes only a single 20-kDa protein in cytoskeletons of O. marina. Functional and phylogenetic aspects of centrin-containing structures in dinoflagellates are discussed.     

Effects of calcium on the longituindal flagellum of Oxyrrhis marina

Summary— 2–4 nm filaments represent a new class of cytoskeletal components. They are found in ciliary and flagellar roots and centrosomes of all eucaryotes. They are also the major components of paraflagellar rods (PFR) in Euglena, trypanosomes and dinoflagellates. Oxyrrhis marina, a marine dinoflagellate, possesses a transverse and a longitudinal flagellum. Only the longitudinal flagellum carries the PFR along the proximal two-thirds of its length. This flagellum is not only capable of the classic flagellar beat but is also able to retract and bend, a property mediated by external calcium. To determine if calcium has a direct role in the bending, experimental conditions were established to permeabilization and reactivation. Our conditions to reactivate the axoneme function (wave propagation) appear similar to those observed in the case of the sea urchin sperm. The results show that in vitro, an increase in calcium concentration induces a conformational change of the longitudinal flagellum in the absence of ATP with a half maximum effect at 0.1 μM. In the presence of ATP, this morphology modification causes a total inhibition of the wave propagation which is replaced by non-propulsive contractions of low amplitude. As these properties are not shared by reactivated sea urchin sperm flagella or the transverse flagellum of O marina devoid of PFR, we propose that PFR are responsible for the bending phenomenon. A calcium shock also induces flagellar excision with a half maximum effect at 0.3 μM, and immunofluorescence results suggest that a centrin-like protein is present in O marina and is responsible for this excision.     

THE CORTICAL MICROTUBULAR CYTOSKELETON OF OXYRRHIS MARINA (DINOPHYCEAE) OBSERVED WITH IMMUNOFLUORESCENCE AND ELECTRON MICROSCOPY1

The cortical microtubular cytoskeleton of Oxyrrhis marina Dujardin was investigated using indirect immunofluorescence and transmission electron microscopy. The cortical microtubular cytoskeleton is unlike that of other previously examined dinoflagellates because all cortical microtubules are oriented longitudinally and do not attach or abut tranverse microtubular arrays. This difference is considered along with other morphological and cytological variables as indicative of Oxyrrhis's phylogenetic position relative to the Dinophyceae.     

Intraspecific selectivity,compensatory feeding and flexible homeostasis in the phagotrophic flagellate <Emphasis Type="Italic">Oxyrrhis marina</Emphasis>: three ways to handle food quality fluctuations

The phagotrophic flagellate Oxyrrhis marina shows a strong stoichiometric plasticity when fed differently grown Rhodomonas salina. We tested whether differently pre-conditioned O. marina displayed selective feeding behaviour from a mixture of nitrogen and phosphorus depleted R. salina. We observed selective feeding of O. marina, always selecting phosphorus rich R. salina independent of the pre-conditioning of the protists. In a second experiment, O. marina was again pre-conditioned either with nitrogen- or phosphorus-depleted R. salina and was refed with either of the differently limited R. salina in single food treatments (not in a mixture). The phagotrophic flagellate displayed compensatory feeding which means that O. marina feed more on the food source which they were not given before. Due to its stoichiometric plasticity, O. marina might handle bad quality food by following the stoichiometry of its prey and additionally by active selective feeding towards P-rich algae to enhance growth. Post-ingestion selection might as well be an important feature which means that ingested elements in excess are quickly excreted and scarce elements are ingested through accelerated food uptake.     

Molecular Characterization of Low-Molecular-Weight Component Protein,Flp, in Actinobacillus actinomycetemcomitans Fimbriae

Fimbriae preparation from Actinobacillus actinomycetemcomitans was found to contain an abundant low-molecular-weight protein (termed Flp) with an apparent molecular mass of approximately 6.5 kDa, in addition to a small amount of 54-kDa protein. Immunogold electron microscopy localized the Flp protein at the bacterial fimbriae but not at the cell surface. The DNA fragment including the flp gene was cloned from A. actinomycetemcomitans 304-a and its nucleotide sequence was determined. An open reading frame of the flp gene was composed of 225 bp encoding a protein of 75 amino acids. Comparison of the translated amino acid sequence with the sequence of native Flp determined by Edman degradation indicated that the N-terminal part of 26 amino acids is leader peptide. The N-terminal sequence of mature Flp exhibited some similarity to type-IV pilin. Furthermore, the processing site of premature Flp is also similar to that of type-IV prepilin, and a gene encoding a protein homologous to type-IV prepilin-like protein leader peptidase was found downstream of the flp gene. These findings indicate that Flp is the major component protein of A. actinomycetemcomitans fimbriae.     

Flagellar scales in Oxyrrhis marina Dujardin

Oxyrrhis marina Dujardin possesses imbricated scales of a tight spiral form on both flagella. These and the associated flagellar hairs are described in detail.     

Biochemical and Immunological Analyses of the Flagellin of Clostridium tyrobutyricum ATCC 25755

The monoclonal antibody 21E7-B12 (IgG₃) can be used in a direct method of Clostridium tyrobutyricum detection based on an immunoenzymatic assay. Immunoelectron microscopy demonstrated that the 21E7-B12 antibody recognized the surface-exposed epitopes on the flagellar filaments of C. tyrobutyricum. After flagellar extraction, the purified flagellin showed an apparent molecular mass of 46 kDa with an isoelectric point of 3.6. Sugar staining, mild periodate oxidation and é-elimination experiments showed that the flagellin was glycosylated and that the 21E7-B12 epitope was located in the sugar moiety. Amino acid composition showed that the flagellar filament protein contained a high percentage of serine and threonine, while proline was absent. The first 23 residues of the N-terminal were determined and sequence homology with other flagellins was found.     

Further nutritional studies on the marine rotifer Encentrum linnhei

Evidence is given that the colourless flagellate Oxyrrhis marina is a natural source of tryptophan in the diet of the rotifer Encentrum linnhei, which inhabits coastal rockpools in western Scotland. The rotifer feeds on this flagellate in addition to the photosynthetic alga Brachiomonas submarina. This study also shows that tryptophan excreted by one alga can be taken up by another and made available to Encentrum, a process which allows the rotifer to grow on combinations of algae which, individually, are nutritionally deficient.     

Ichthyotoxicity of Chattonella marina (Raphidophyceae) to damselfish (Acanthochromis polycanthus): the synergistic role of reactive oxygen species and free fatty acids

This investigation aimed to elucidate the relative roles of putative brevetoxins, reactive oxygen species and free fatty acids as the toxic principle of the raphidophyte Chattonella marina, using damselfish as the bioassay. Our investigations on Australian C. marina demonstrated an absence or only very low concentrations of brevetoxin-like compounds by radio-receptor binding assay and liquid chromatography–mass spectroscopy techniques. Chattonella is unique in its ability to produce levels of reactive oxygen species 100 times higher than most other algal species. However, high levels of superoxide on their own were found not to cause fish mortalities. Lipid analysis revealed this raphidophyte to contain high concentrations of the polyunsaturated fatty acid eicosapentaenoic acid (EPA; 18–23% of fatty acids), which has demonstrated toxic properties to marine organisms. Using damselfish as a model organism, we demonstrated that the free fatty acid (FFA) form of EPA produced a mortality and fish behavioural response similar to fish exposed to C. marina cells. This effect was not apparent when fish were exposed to other lipid fractions including a triglyceride containing fish oil, docosahexaenoate-enriched ethyl ester, or pure brevetoxin standards. The presence of superoxide together with low concentrations of EPA accelerated fish mortality rate threefold. We conclude that the enhancement of ichthyotoxicity of EPA in the presence of superoxide can account for the high C. marina fish killing potential.     

The occurrence of body scales in Oxyrrhis marina Dujardin

Previously reported flagellar scales of spiral form on Oxyrrhis marina Dujardin extend to cover the whole body. These and the distribution of trichocysts are described in detail.     

A comparative approach to study inhibition of grazing and lipid composition of a toxic and non-toxic clone of Chrysochromulina polylepis (Prymnesiophyceae)

Since the massive bloom in 1988 in the North Sea, the prymnesiophyte flagellate Chrysochromulina polylepis Manton et Parke has been known for its ichtyotoxicity. Laboratory experiments using two different clones of C. polylepis were conducted in a comparative approach. Both clones were nearly similar in size and shape, but differed in their toxicity, as demonstrated by the Artemia bioassay. In order to study the effects of toxic C. polylepis on protozooplankton grazers, grazing experiments were performed with the heterotrophic dinoflagellate Oxyrrhis marina Dujardin as grazer. A first experiment was carried out in order to follow batch culture growth and initial grazing of O. marina when fed toxic or non-toxic clones of C. polylepis. Ingestion of the toxic clone was 27% of ingestion when fed with the non-toxic clone. When O. marina was fed with the toxic clone, vacuoles within O. marina contained fewer food particles per cell and the cells attained slower division rate (58% of the division rate estimated for the non-toxic clone). A second experiment was conducted to determine the grazing and growth response of O. marina as a function of algal food concentration. Profound differences in ingestion, clearance, division and gross growth efficiency of O. marina when fed the two clones of C. polylepis again were apparent. However, even at algal concentrations of 400×10³ ml⁻¹, O. marina is not killed by the presence or by the ingestion of toxic C. polylepis, indicating that the toxin deters grazers. In addition to grazing experiments, lipid classes and fatty acids of both algal clones were analysed and compared in order to follow the hypothesis that toxicity of C. polylepis is caused by liposaccharides, lipids, or fatty acids. However, the chemical composition with respect to lipid classes and fatty acids of both clones were quite similar, making an involvement of these substances in the toxicity towards Artemia and O. marina unlikely.     

Immunogold-localization and synthesis of an oil-body membrane protein in developing soybean seeds

The synthesis of a major oil-body membrane brotein was studied in maturing soybean (Glycine max (L.) Merr.) cotyledons. The membrane contained four abundant proteins with apparent molecular mass (M_r) of 34000, 24000, 18000 and 17000. The M_r=24000 protein (mP 24) was selected for more detailed analysis. The protein was purified to apparent homogeneity by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and isolated from the gel by electroelution or chemical hydrolysis of gel crosslinks. It was then used to elicit rabbit antibodies which were judged to be specific when assayed by SDS-PAGE-immunoblot procedures. The mP 24 was localized in immature soybean cotyledon cells by indirect immunogold procedures on thin sections of Lowicryl- and LR-White-embedded tissue. Indirect labeling with the primary antiserum followed by colloidal gold-protein A showed specific labeling of the oil-body membrane and an absence of label on the other subcellular organelles including the endoplasmic reticulum (ER). Parallel tissue samples were studied by conventional transmission electron microscopy. Although segments of the ER were observed to be closely juxtaposed to the oil bodies, continuity between the two organelles was not observed. The synthesis of mP 24 was studied by in-vitro translation and in-vivo labeling with [³H]leucine followed by indirect immunoaffinity isolation of the labeled products. The SDS-PAGE fluorography results indicated that the primary translation product and the in-vivo synthesized protein have the same M_r, and this is also the same M_r as the protein in the mature membrane.Abbreviations and symbols DATD N N'-diallyltartardiamide - EM electron microscopy/scopic - ER endoplasmic reticulum - IgG immunoglobulin G - M_r apparent molecular mass - PBS phosphate-buffered saline - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TBS Trisbuffered saline     

A UNIQUE MITOTIC VARIATION IN THE MARINE DINOFLAGELLATE OXYRRHIS MARINA (PYRROPHYTA)1

Mitosis is described in the flagellate Oxyrrhis marina Dujardin and is compared in related genera. Dense plaques develop in the nuclear envelope at prophase and give rise to an intranuclear spindle. Some of the microtubules associate with the chromosomes while others extend across the nucleus. The basal bodies migrate toward the poles early in division and retain a position lateral to the nuclear poles throughout mitosis. Microtubules are not present between the nucleus and the basal bodies. The nucleolus is persistent and elongates throughout anaphase and telophase. Chromosomal separation is accomplished by sliding of non-chromosomal microtubules and by elongation of the nuclear envelope rather than by shortening of the spindle microtubules. The nuclear envelope begins to constrict in the center early in anaphase. Continued constriction of the envelope and elongation of the nucleus leads to the formation of a dumbbell-shaped nucleus by late telophase. Mitosis culminates by the constriction of the nucleus into two daughter nuclei. The taxonomic position of Oxyrrhis marina is discussed in light of these findings.     

CHEMOSENSORY CAPABILITIES IN THE PHAGOTROPHIC DINOFLAGELLATE GYMNODINIUM FUNGIFORME11

The non-photosynthetic, phagotrophic dinoflagellate, Gymnodinium fungiforme Anissimova is attracted to a variety of amino acids and other organic compounds. Glycine, taurine and serine attracted the dinoflagellates at a threshold detection level of 10^?8 M fallowed by dextrose (10^?7 M) and alanine, proline and threonine (10^?6 M). Glycine, taurine and alanine are three of the most abundant free amino acids found in invertebrates and protozoa which are major food sources of this dinoflagellate. Three additional species of cultured heterotrophic dinoflagellates were exposed to the water soluble fraction of a shrimp extract known to attract G. fungiforme. All three species responded to the extract, but one species, Oxyrrhis marina, did so only after changing its food source. It is suggested that chemosensory behavior may be suppressed or expressed depending on culture conditions.     

High salinity reduces the content of a highly abundant 23-kDa protein of the mangrove<Emphasis Type="Italic"> Bruguiera parviflora</Emphasis>

A significant decrease in the amount of a protein, whose migration in two-dimensional gel electrophoresis corresponds to an apparent molecular mass of 23 kDa and pI=6.5, was observed in leaves of NaCl-treated Bruguiera parviflora (Roxb.) Wt. & Arn. ex Griff. seedlings. This particular salt-sensitive protein, designated as SSP-23, almost disappeared after 45 days of treatment in 400 mM NaCl as compared to untreated seedlings (0 mM NaCl) where the presence of the protein was significant. A polyclonal antibody raised against the 23-kDa protein was used to determine the subcellular localization of this protein in leaves by cross-reaction with proteins from isolated chloroplasts, mitochondria, peroxisomes and cytosol fractions on Western blots. SSP-23 was confirmed to be localized in the cytosol by immunoblotting. The disappearance of SSP-23 as a result of high NaCl treatment suggests that this protein is salt-sensitive and has a possible role in salt adaptation.     

Nitric oxide mediates oxylipin production and grazing defense in diatoms

Diatom blooms are important features of productive marine ecosystems and are known to support higher trophic levels. However, when stressed or wounded, diatoms can produce oxylipin molecules known to inhibit the reproduction and development of copepods and decrease microzooplankton growth rates. Using oxylipin chemical treatments, lipidomic analysis and functional genomic approaches, we provide evidence that nitric oxide (NO) and oxylipin signalling pathways in diatoms respond to protist grazers, resulting in increased defence fitness and survival. Exposure of the diatom Phaeodactylum tricornutum to the dinoflagellate Oxyrrhis marina resulted in NO production by P. tricornutum and pronounced change in its dissolved oxylipin profile. Experimentally elevating levels of NO also resulted in increased oxylipin production, and lower overall grazing rates. Furthermore, O. marina preferentially grazed on P. tricornutum prey with lower levels of NO, suggesting that this molecule and its effect on oxylipin pathways play a key role in prey selection. Exposure of O. marina grazing on P. tricornutum to exogenous oxylipins also decreased grazing rates, which is consistent with a grazing deterrence role for these molecules. These results suggest that NO and oxylipin production help to structure diatom communities, in part by modulating interactions with microzooplankton predators.     

Conceptual Bases for Prey Biorecognition and Feeding Selectivity in the Microplanktonic Marine Phagotroph <Emphasis Type="Italic">Oxyrrhis marina</Emphasis>

It is suspected that phagotrophic marine protozoa might possess feeding receptors that enable them to discern the nutritional quality of individual prey items (during prey-handling) on the basis of their cell-surface biochemistry. This article reviews advances in our understanding of the molecular mechanisms that mediate the biorecognition and selection of nonself (microalgal) prey items by the microplanktonic marine phagotroph Oxyrrhis marina. The potential importance of lectin–glycan interactions is first considered in view of findings which demonstrate that O. marina possesses lectin-like feeding receptors specific for prey-surface (mannose) glycoconjugates. Secondly, some conceptual bases for indirect or ‘opsonic’ modes of prey biorecognition mediated by soluble prey-labelling proteins are presented. Finally, the possibility that some accounts of selective feeding in O. marina might result from the noxious effects of prey-associated chemicals rather than active ‘distaste’ by phagotrophic cells is discussed. Recent evidence for toxic superoxide (O₂ ⁻) production by marine microalgae is afforded particular attention given that release of O₂ ⁻ anions can be exacerbated by the binding of mannose-specific lectins to the microalgal cell wall; a novel model for grazing-activated chemical defence is proposed.     

Identification and partial purification of DnaK homologue from extremely halophilic archaebacteria, Halobacterium cutirubrum

The levels of synthesis of six proteins were increased at elevated growth temperature of the extremely halophilic archaebacterium Halobacterium cutirubrum. One of these proteins, with an apparent molecular mass of 97 kDa on sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS–PAGE), bound to an ATP-agarose column in the presence of 4 M NaCl, but not in the absence of salt, indicating that this protein retained its ATP-binding activity only at high salt concentration. The NH₂-terminal sequence of this protein and the internal sequences of the tryptic peptides covering 1/3 of the total number of residues coincided with that deduced from the nucleotide sequence of the dnaK gene isolated from H. cutirubrum. The results strongly suggest that this apparent 97-kDa protein is the gene product of dnaK, although the molecular mass calculated from the nucleotide sequence is only 68,495, much smaller than the value of this protein determined by SDS–PAGE. Ferguson plot analysis indicated that this protein showed anomalous mobility on SDS–PAGE. We have purified DnaK homologue to greater than 90% homogeneity with stepwise elution from an ATP-agarose column.     

Molecular and biochemical characterization of a fructose-6-phosphate-forming and ATP-dependent fructokinase of the hyperthermophilic archaeon <Emphasis Type="Italic">Thermococcus litoralis</Emphasis>

Close to an operon encoding an ABC transporter for maltose and trehalose, Thermococcus litoralis contains a gene whose encoded sequence showed similarity to sugar kinases. We cloned this gene, now called frk, and expressed it as a C-terminal His-tag version in Escherichia coli. We purified the recombinant protein, identified it as an ATP-dependent and fructose-6-phosphate-forming fructokinase (Frk) and determined its biochemical properties. At its optimal temperature of 80°C, the apparent K_m and V_max values of Frk were 2.3 mM and 730 U/mg protein for fructose at saturating ATP concentration, and 0.81 mM and 920 U/mg protein for ATP at saturating fructose concentration. The enzyme did not lose activity at 80°C for 4 h. Under denaturating conditions in SDS-PAGE, it exhibited a molecular mass of 35 kDa. Gel-filtration chromatography revealed a molecular mass of 58 kDa, indicating a dimer under nondenaturating, in vitro conditions.