Kanae Koike

The d-galactose-binding lectin of the octocoral Sinularia lochmodes: characterization and possible relationship to the symbiotic dinoflagellates

A D-galactose binding lectin (SLL-2) was isolated from Sinularia lochmodes, an octocoral, by a combination of affinity chromatography on acid-treated agarose and FPLC on Superdex 200. SLL-2 agglutinated rabbit and horse erythrocytes while SLL-1, a minor component, reacted only with rabbit erythrocytes. SLL-2 is a glycoprotein with a molecular mass of 122 kDa and is composed of eight identical subunits (15 kDa). The sequence of the amino terminal region of SLL-2 did not show any apparent homology to the sequences of other animal and plant lectins. D-Galactose, N-acetyl-D-galactosamine, lactose, and melibiose were moderate inhibitors to the agglutination of rabbit erythrocytes. In contrast, horse erythrocytes were much more susceptible to agglutination by SLL-2, which was inhibited by sugars and glycoproteins such as D-galactose, N-acetyl-D-galactosamine, lactose, melibiose, and porcine stomach mucin. SLL-2 showed considerable tolerance to heating and kept its activity after heating at 80 degrees C for 60 min. In immuno-histochemical studies using an anti-SLL-2 antiserum and protein A gold conjugate, SLL-2 was found to be present in high amounts in the nematocysts. SLL-2 was also detected on the surface of symbiotic dinoflagellate, Symbiodinium sp. cells irrespective whether they were surrounded with or without host cells. These observations suggest the presence of lectin-mediated interaction between symbiotic dinoflagellates and S. lochmodes.

Isolation and Characterization of a Single-Stranded DNA Virus Infecting Chaetoceros lorenzianus Grunow

ABSTRACT Diatoms are one of the most significant primary producers in the ocean, and the importance of viruses as a potential source of mortality for diatoms has recently been recognized. Thus far, eight different diatom viruses infecting the genera Rhizosolenia and Chaetoceros have been isolated and characterized to different extents. We report the isolation of a novel diatom virus (ClorDNAV), which causes the lysis of the bloom-forming species Chaetoceros lorenzianus, and show its physiological, morphological, and genomic characteristics. The free virion was estimated to be ∼34 nm in diameter. The arrangement of virus particles appearing in cross-section was basically a random aggregation in the nucleus. Occasionally, distinctive formations such as a ring-like array composed of 9 or 10 spherical virions or a centipede-like array composed of rod-shaped particles were also observed. The latent period and the burst size were estimated to be <48 h and 2.2 × 104 infectious units per host cell, respectively. ClorDNAV harbors a covalently closed circular single-stranded DNA (ssDNA) genome (5,813 nucleotides [nt]) that includes a partially double-stranded DNA region (979 nt). At least three major open reading frames were identified; one showed a high similarity to putative replicase-related proteins of the other ssDNA diatom viruses, Chaetoceros salsugineum DNA virus (previously reported as CsNIV) and Chaetoceros tenuissimus DNA virus. ClorDNAV is the third member of the closed circular ssDNA diatom virus group, the genus Bacilladnavirus.

Evidence of phagotrophy in Dinophysis fortii (Dinophysiales, Dinophyceae), a dinoflagellate that causes diarrhetic shellfish poisoning (DSP)

Food vacuoles were found in one species of pho‐totrophic Dinophysis, Dinophysis fortii Pavillard, collected in Okkirai Bay. Under transmission electron microscopy, almost 70% of observed food vacuoles were characterized by membranous profiles and contained large numbers of mitochondria. The mitochondria in the food vacuole had different morphologies from those in the D. fortii cytoplasm. This indicates that these vacuoles are not autolytic accumulation bodies, but ‘true’ food vacuoles. Identification of the origin of the contents failed, but the existence of large amounts of foreign mitochondria implies that the contents in the vacuoles were derived from eukaryotic prey. Other than the observation of the food vacuoles, bacterial cells were observed in the flagellar canal. Because the flagellar canal and connecting pusule sacs had been reported to relate to macromolecule uptake, the prey organisms of D. fortii were assumed to be both eukaryotic and prokaryotic organisms.

Cellular Origin of Dysiherbaine, an Excitatory Amino Acid Derived from a Marine Sponge

The cellular origin of dysiherbaine, a marine‐sponge toxin, was investigated immunohistochemically by using an anti‐dysiherbaine antibody. Dysiherbaine‐like immunoreactivity was found to be localized in spherical cells harbored in the sponge mesohyl. A combination of ribosomal RNA gene (rDNA) analysis and cell‐morphology analysis revealed that the spherical cells were Synechocystis cyanobacteria. However, the sponge, identified as Lendenfeldia chondrodes on the basis of its rDNA sequence, appeared to contain two different chemotypes—dysiherbaine‐producing (DH+) and nondysiherbaine‐producing (DH−)—both of which inhabited the same region. Synechocystis cells in the DH− sponge were not labeled with antibody, although the 16S rDNA gene profile of the cyanobacteria in the DH− sponge was indistinguishable from that of the cyanobacteria in the DH+ sponge. On the basis of these results, we hypothesize that dysiherbaine is a metabolite of certain varieties of endosymbiotic Synechocystis sp.

Cyanobacterial endosymbionts in the benthic dinoflagellate Sinophysis canaliculata (Dinophysiales, Dinophyceae).

Photosynthetic dinoflagellates possess a great diversity of plastids that have been acquired through successful serial endosymbiosis. The peridinin-containing plastid in dinoflagellates is canonical, but many other types are known within this group. Within the Dinophysiales, several species of Dinophysis contain plastids, derived from cryptophytes or haptophytes. In this work, the presence of numerous intracellular cyanobacteria-like microorganisms compartmentalized by a separate membrane is reported for the first time within the benthic dinophysoid dinoflagellate Sinophysis canaliculata Quod et al., a species from a genus morphologically close to Dinophysis. Although the contribution of these cyanobacterial endosymbionts to S. canaliculata is still unknown, this finding suggests a possible undergoing primary endosymbiosis in a dinoflagellate.

Cellular and subcellular localization of kainic acid in the marine red alga Digenea simplex

Polyclonal antibodies specific for the excitatory amino acid, kainic acid (KA), were raised in rabbits. The antibody recognized KA but did not cross-react with other structurally related amino acids, including glutamate. We used this anti-KA antibody to localize KA immunohistochemically in the KA-producing red alga Digenea simplex. KA immunoreactivity was most dense in the fine cylindrical thallus, which covers the middle to upper part of the alga. The cortical cells, but not the inner layers of the main axis, and cells of the rhizoid were also stained with this antibody. The presence of KA in cells that cover the surface of the alga might reflect its role in chemical defense. At the subcellular level, KA immunoreactivity was most intense in the nucleus, pit plugs, and the electron-dense areas denoted as “granule bodies”, which were found only in the pericentral cells of the thallus.

MECHANISM OF GAMETE FUSION IN DINOPHYSIS FORTII (DINOPHYCEAE, DINOPHYTA): LIGHT MICROSCOPIC AND ULTRASTRUCTURAL OBSERVATIONS1

A variety of studies have examined the sexual life cycle of species belonging to the genus Dinophysis Ehrenberg. Here, we used TEM to investigate the mechanism of cellular fusion during the sexual life cycle in Dinophysis fortii Pavillard. We observed that fusion always occurred between a normal‐sized cell and a small cell following attachment of their ventral margins. After cell attachment, the small cell moved toward the epitheca of the normal‐sized cell, and the cingular and sulcal lists of the small cell shrunk or were almost lost. The epitheca of the normal‐sized cell then opened between the cingulum plates and the upper cingular list, after which the small cell was gradually engulfed. This is the first ultrastructural observation in a dinoflagellate of a larger cell opening its epitheca to engulf the smaller gamete. In another case, the normal‐sized cell did not open the epitheca, the cell wall of the attached small cell underwent extensive extracellular digestion, and the cytoplasm appeared to flow into the normal‐sized cell via the periflagellar area. Inflow of the nucleus was not observed in this case, suggesting that it represented a failure of sexual fusion. In both cases, membranous separations between the cytoplasm of the two cells were not observed. At the beginning of the fusion process, the nucleus of the small cell was substantially deformed. The planozygote, formed upon completion of sexual fusion, sometimes had two longitudinal flagella, but was identical to a normal vegetative cell in its cellular shape, as already mentioned by previous authors.

Mechanisms of Maternal Inheritance of Dinoflagellate Symbionts in the Acoelomorph Worm Waminoa litus

Waminoa litus is a zooxanthella-bearing acoel worm that infests corals. It is unique to Bilateria in that it transmits its algal symbionts vertically via eggs irrespective of the heterogeneity of the symbionts. It simultaneously harbors two dinoflagellate genera: Symbiodinium and Amphidinium. In this study, we examined the timing and vertical transmission pathway of algal symbionts in W. litus using light and electron microscopy. The oogenesis of the worm can be divided into three stages: stage I, in which the ovary is absent; stage II, the early vitellogenic zone containing immature oocytes formed in the ovary; and stage III, with both early and late vitellogenic zones in the body. In the early vitellogenic zone at stage II, oocytes are surrounded by accessory-follicle cells (AFCs). Both Symbiodinium and Amphidinium symbionts are not initially observed in the oocytes, but are observed in the AFCs. In the late vitellogenic zone at stage III, oocytes are enveloped by a complete sheath of AFCs; the algal symbionts are taken up by the late vitellogenic oocytes. These observations suggest that AFCs mediate the transfer of the algae from the parent to the oocytes. Ribotype analyses of the Symbiodinium symbionts revealed that they differ from those harbored by coral in the same experimental aquarium. These results indicate that W. litus has an active algal transport pathway and maintains a specific lineage of Symbiodinium via vertical transmission.

The impact of the overexpression of human UDP-galactose transporter gene hUGT1 in tobacco plants.

When the human UDP-galactose transporter 1 gene (hUGT1) was introduced into tobacco plants, the plants displayed enhanced growth during cultivation, and axillary shoots had an altered determinate growth habit, elongating beyond the primary shoots and having a sympodial growth pattern similar to that observed in tomatoes at a late cultivation stage. The architecture and properties of tissues in hUGT1-transgenic plants were also altered. The leaves had an increase in thickness, due to an increased amount of spongy tissue, and a higher content of chlorophyll a and b; the stems had an increased number of xylem vessels and accumulated lignin and arabinogalactan proteins (AGPs). Some of these characteristics resembled a gibberellin (GA)-responsive phenotype, suggesting involvement of GA. RT-PCR-based analysis of genes involved in GA biosynthesis suggested that the GA biosynthetic pathway was not activated. However, an increase in the proportion of galactose in polysaccharide side chains of AGPs was detected. These results suggested that because of higher UDP-galactose transport from the cytosol to the Golgi apparatus, galactose incorporation into polysaccharide side chains of AGP is involved in the gibberellin response, resulting in morphological and architectural changes.

Bothrosome Formation in Schizochytrium aggregatum (Labyrinthulomycetes, Stramenopiles) during Zoospore Settlement

Labyrinthulomycetes are characterized by the presence of ectoplasmic nets originating from an organelle known as the bothrosome, whose evolutionary origin is unclear. To address this issue, we investigated the developmental process from a zoospore to a vegetative cell in Schizochytrium aggregatum. After disappearance of the flagellum during zoospore settlement, the bothrosome emerged at the anterior-ventral pole of the cells. A new Golgi body also appeared at this stage, and the bothrosome was positioned close to both the new and the old Golgi bodies. This observation suggested that the Golgi body is related to the formation of the bothrosome. Actin appeared as a spot in the same location as the newly appeared bothrosome, as determined by immunofluorescence labeling. An immunoelectron microscopic analysis revealed that actin was present in the ectoplasmic nets and in the cytoplasm around the bothrosome, indicating that the electron-dense materials of the bothrosome are not the polar center of F-actin. This suggests that actin filaments pull the endoplasmic reticulum to the bothrosome and induce the membrane to become evaginated within ectoplasmic nets.