Molecular Phylogeny of the Benthic Dinoflagellate Genus Amphidiniopsis and its Relationships with the Family Protoperidiniaceae.

The genus Amphidiniopsis is a benthic (sand-dwelling) lineage of thecate dinoflagellates, containing 19 morphologically diverse species. Past work has shown that some Amphidiniopsis species form a clade with the sand-dwelling Herdmania litoralis as well as some planktonic species in the family Protoperidiniaceae (i.e. the Monovela group). Still, our contemporary knowledge regarding Amphidiniopsis is limited, compared to the Protoperidiniaceae. To this end, we obtained 18S rDNA data from seven Amphidiniopsis species and a part of the 28S rDNA from four Amphidiniopsis species, with the goal of improving our understanding of phylogenetic relationships among Amphidiniopsis and the Monovela group. Results from the molecular phylogenetic analyses showed that Amphidiniopsis spp., with the exception of A. cf. arenaria, H. litoralis, and members within the Monovela group formed a single clade. Within the clade, relationships among Amphidiniopsis spp. and the Monovela group were more complicated - some subclades contained both representatives of Amphidiniopsis and the Monovela group. Our study suggests that habitat (benthic or planktonic), as well as traditionally used, general morphological characteristics, do not reflect molecular phylogenetic relationships, and that the taxonomy of the sand-dwelling genus Amphidiniopsis, and the planktonic family Protoperidiniaceae, should be reconsidered simultaneously.

Chlorophycean parasite on a marine fish, Sillago japonica (Japanese sillago).

A green spotted Japanese sillago (Sillago japonica) was caught by a fisherman and brought to the laboratory for pathological inspection. The green spots were abundant on the lateral line and more extensively so within the mouth cavity. In both sites, green spots were embedded within the fish flesh and formed 2-3mm dome-shaped colonies. SEM revealed these colonies to harbor numerous unknown cells with small, surface warts (ornamentations). Molecular analysis showed the cells were Desmodesmus (D. komarekii), a common freshwater coccoid green alga found in ponds and rivers worldwide. It is uncertain how the host fish came to be infected with the alga which was not merely attached externally but embedded within the flesh and inside the mouth cavity. This is the first case of parasitic form of coccoid green algae in marine fish and provides new insights into the variable nature of green algae.

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.

[Toxicity and toxin profile of the dinoflagellate Alexandrium tamiyavanichii and toxic mussels in Harima-Nada of Seto Inland Sea, Japan].

From October to November 2004, the paralytic shellfish poison (PSP)-producing dinoflagellate Alexandrium tamiyavanichii was observed at Harima-Nada, Seto Inland Sea at a maximum cell density of 4,960 cells/L. The wild cells of the dinoflagellate collected from the same seawaters, and cultured cells derived from them showed toxicity scores of 6.25-15.4 x 10(-4) and 2.7-3.5 x 10(-4) MU/cell, respectively, both of which were much higher than those of previously reported strains. PSP of the wild cells was mainly composed of gonyautoxin (GTX) 5 (40.6-52.4 mol%) and GTX4 (15.6-24.8 mol%), showing a unique composition that was greatly different from those of the previously reported strains, or of the cultured cells, whose main toxin component was GTX3 (average 37.6 mol%). The mussel Mytilus galloprovincialis collected from the same area in the same period accumulated a relatively high level of PSP (13-28 MU/g), suggesting a risk that A. tamiyavanichii may induce high-level PSP contamination of bivalves even at a cell density as low as around 5,000 cells/L.

Mitochondrial group II introns in the raphidophycean flagellate Chattonella spp. suggest a diatom-to-Chattonella lateral group II intron transfer.

In the cytochrome c oxidase subunit I (cox1) gene of four raphidophycean flagellates Chattonella antiqua, C. marina, C. ovata, and C. minima we found two group II introns described here as Chattonella cox1-i1 and Chattonella cox1-i2 encoding an open reading frame (ORF) comprised of three domains: reverse transcriptase (RT), RNA maturase (Ma) and zinc finger (H-N-H) endonuclease domains. The secondary structures show both Chattonella cox1-i1 and Chattonella cox1-i2 belong to group IIA1, albeit the former possesses a group IIB-like secondary structural character in the epsilon' region of arm I. Our phylogenetic analysis inferred from RT domain sequences of the intronic ORF, comparison of the insertion sites, and the secondary structures of the introns suggests that Chattonella cox1-i1 likely shares an evolutionary origin with the group II introns inserted in cox1 genes of five phylogenetically diverged eukaryotes. In contrast, Chattonella cox1-i2 was suggested to bear a close evolutionary affinity to the group II introns found in diatom cox1 genes. The RT domain-based phylogeny shows a tree topology in which Chattonella cox1-i2 is nested in the diatom sequences suggesting that a diatom-to-Chattonella intron transfer has taken place. Finally, we found no intron in cox1 genes from deeper-branching raphidophyceans. Based on parsimonious discussion, Chattonella cox1-i1 and Chattonella cox1-i2 have invaded into the cox1 gene of an ancestral Chattonella cell after diverging from C. subsalsa.

Yessotoxin analogues in several strains of Protoceratium reticulatum in Japan determined by liquid chromatography-hybrid triple quadrupole/linear ion trap mass spectrometry.

Several strains of Protoceratium reticulatum, one of the dinoflagellates producing yessotoxins (YTXs), were collected from various shellfish producing areas in Japan. YTXs in the cultured strains were analyzed by liquid chromatography-mass spectrometry (LC-MS). Neutral loss scan monitoring, multiple reaction monitoring (MRM) for more than 20 YTX analogues, and full-scan MS/MS spectra obtained with a hybrid triple quadrupole/linear ion trap mass spectrometer showed that yessotoxin (YTX), 45,46,47-trinoryessotoxin (trinorYTX), 1-homoyessotoxin (homoYTX), and 45,46,47-trinor-1-homoyessotoxin (trinor-1-homoYTX) were the dominant toxins in these strains of P. reticulatum. Enone isomer of 42,43,44,45,46,47,55-heptanor-41-oxoyessotoxin (noroxoYTX enone) was also detected in some strains. Toxin profiles and contents were different among the strains. Some strains produced YTX, trinorYTX, 1-homoYTX, trinor-1-homoYTX, and noroxoYTX enone, whereas other strains produced only YTX or 1-homoYTX. This is the first identification of 1-homoYTX and noroxoYTX enone in P. reticulutum in Japan. Some strains did not produce any detectable YTX analogues.

First paralytic shellfish poison (PSP) infestation of bivalves due to toxic dinoflagellate Alexandrium tamiyavanichii, in the southeast coasts of the Seto Inland Sea, Japan.

The mussel Mytilus edulis and the cultured ark shell Anadara broughtonii in the southeast coasts of the Seto Inland Sea were contaminated with paralytic shellfish poison (PSP) following the appearance of the dinoflagellate Alexandrium tamiyavanichii in early December 1999. A. tamiyavanichii plankton collected around the Straits of Naruto on December 3, 1999 showed PSP toxicity, of which 83 mol% was accounted for by GTX2, GTX3 and GTX4. Its specific toxicity was 112.5 fmol/cell, and one MU was equivalent to 7,200 cells. Toxicity values at the beginning of toxification were 4.7 MU/g for the ark shell and 7.3 MU/g for the mussel. In the former, the value remained at almost 4 MU/g, resulting in prohibition of marketing for about two months. In the latter, it sharply decreased to less than 4 MU/g. These bivalves collected during the toxification period were dissected into five tissues, mantle, adductor muscle, hepatopancreas, gills and "others", and submitted to high-performance liquid chromatography (HPLC). The cultured ark shell accumulated GTX2, GTX3 and STX as major components and GTX1, GTX4, GTX5, neoSTX, dcSTX and PX1-3 (C1-C3) as minor ones. The amount of GTX3 decreased with time, while STX tended to increase. At the early stage of PSP toxification, toxins were accumulated in the gills and "others", most of which were quickly detoxified. On the other hand, PSP of the toxified mussel consisted of GTX4 as a main component, and GTX1, GTX2, GTX3, GTX5, STX and PX1-2 (C1-C2) as minor ones. Its toxin composition pattern was similar to that of the ingested causative plankton. Its total toxin decreased soon after disappearance of the dinoflagellate. During the decrease of toxicity, PSP tended to be retained in the hepatopancreas, resulting in accumulation of 50 mol% of total toxin.