IntroductionIn the dinoflagellate genus Dinophysis, some species are known to cause diarrhetic shellfish poisoning (DSP). The physiological and ecological characteristics of this genus are not yet fully understood due to difficulties in culturing the organisms.Dinophysis caudata Saville-Kent is one of the toxic species that causes DSP. Okadaic acid (OA) and dinophysistoxin-1 (DTX1) were detected from D. caudata cells (Ͻ76.3 pg cell Ϫ1 of OA and DTX1 in total) in Sapian Bay, the Philippines (Marasigan et al. 2001). This species is widely distributed in tropical and temperate waters and can appear abundantly in coastal waters, with a red tide of this species associated with mass mortalities of fish being reported in the Seto Inland Sea, Japan (Okaichi 1967). Holmes et al. (1999) reported that D. caudata was the main species causing DSP in green mussels in Singapore. Thus, D. caudata might be one of the main causative species of DSP in the future, especially in tropical regions. The establishment of cultures is crucial to study the physiology and toxicology of this species. Recently, Park et al. (2006) succeeded in cultivating Dinophysis acuminata Claparède et Lachmann at high cell densities (Ͼ11,000 cell mL Ϫ1 ) and maintained them for a long period (Ͼ6 months) by feeding the ciliate Myrionecta rubra (Lohmann 1908) grown with a cryptophyte Teleaulax sp. In this report, we followed their experimental design, and succeeded in cultivating D. caudata. We report here the conditions necessary for cultivation of D. caudata and describe their feeding strategy. Materials and MethodsThe marine ciliate Myrionecta rubra and the cryptophyte Teleaulax amphioxeia (Conrad) Hill were isolated from Inokushi Bay (131°53ЈE, 34°47ЈN) at the end of February 2007 in Oita Prefecture, Japan. Myrionecta rubra and T. amphioxeia were identified by their morphology and sequence data from the nuclear small subunit rDNA. The sequences are deposited in GenBank under accession num- (2006) succeeded in cultivating the toxic dinoflagellate Dinophysis acuminata and maintaining them by feeding the ciliate Myrionecta rubra grown with a cryptophyte Teleaulax sp. After this report, the present study is the second report of propagation of a Dinophysis species (Dinophysis caudata) under laboratory conditions and describes the maintenance of several clonal strains kept at high abundance (Ͼ5,000 cells mL Ϫ1) for a relatively long period (Ͼ4 months) when fed on M. rubra with the addition of Teleaulax amphioxeia. We confirmed that D. caudata swam actively around its ciliate prey and inserted its peduncle (feeding tube) into the ciliate. Thereafter, the prey became immobile and rounded. Dinophysis caudata actively ingested the cytoplasm of the prey through the peduncle. Dinophysis caudata grew at a growth rate of 1.03 divisions day Ϫ1 when supplied with M. rubra as prey, reaching a maximum concentration of ca. 5,000 cell well Ϫ1 (810 mL) during a 9 day growth experiment. In contrast, a culture of D. caudata was not able to be established in the absence of the...

To determine the process of population expansion and ascertain the origin of the Sea of Japan population, in a noxious red tide forming dinoflagellate Cochlodinium polykrikoides, 13 samples, isolated from 11 different localities in Japanese and Korean coasts, were analysed using 10 polymorphic microsatellites. Analyses by nonmetric multidimensional scaling plots of pairwise F(ST), global amova, and genetic admixture analysis identified three clusters--the Sea of Japan populations, Yatsushiro Sea (Kumamoto Pref.) populations, and other populations--indicating genetic structuring of the 13 samples into three distinct populations. In the proportion of shared alleles by pairwise individuals (P(SAxy)) analyses between the Sea of Japan and the other samples, P(SAxy) was extremely low compared with that among the Sea of Japan or among other samples, indicating that a large genetic barrier has occurred between the populations. No significant relationship of isolation-by-distance patterns and almost no genetic distance were detected between pairwise samples of the Sea of Japan, although there is a maximal distance of > 600 km between samples. In addition, P(SAxy) data among the samples were extremely high compared with those among other samples, clearly showing that a large-scale transfer from west to east has occurred via the Tushima Warm Current. In the P(SAxy) data of the Seto Inland Sea and Pacific samples, individuals showing relatively high P(SAxy) were concentrated in the three areas of Nagasaki, Harima, and Mie, suggesting that frequent transfer may have occurred by human-assisted dispersal, although Nagasaki and Mie are separated by a distance of approximately 700 km.

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We analyzed cryptophyte nucleomorph 18S rRNA gene sequences retained in natural Myrionecta rubra cells and plastid 16S rRNA gene and psbA sequences retained in natural cells of several Dinophysis species collected from Japanese coastal waters. A total of 715 nucleomorph sequences obtained from 134 M. rubra cells and 564 plastid 16S rRNA gene and 355 psbA sequences from 71 Dinophysis cells were determined. Almost all sequences in M. rubra and Dinophysis spp. were identical to those of Teleaulax amphioxeia, suggesting that M. rubra in Japanese coastal waters preferentially ingest T. amphioxeia. The remaining sequences were closely related to those of Geminigera cryophila and Teleaulax acuta. Interestingly, 37 plastid 16S rRNA gene sequences, which were different from T. amphioxeia and amplified from Dinophysis acuminata and Dinophysis norvegica cells, were identical to the sequence of a D. acuminata cell found in the Greenland Sea, suggesting that a widely distributed and unknown cryptophyte species is also preyed upon by M. rubra and subsequently sequestered by Dinophysis. To confirm the reliability of molecular identification of the cryptophyte Teleaulax species detected from M. rubra and Dinophysis cells, the nucleomorph and plastid genes of Teleaulax species isolated from seawaters were also analyzed. Of 19 isolates, 16 and 3 clonal strains were identified as T. amphioxeia and T. acuta, respectively, and no sequence variation was confirmed within species. T. amphioxeia is probably the primary source of prey for M. rubra in Japanese coastal waters. An unknown cryptophyte may serve as an additional source, depending on localities and seasons.The marine dinoflagellate genus Dinophysis comprises photosynthetic and nonphotosynthetic members and is globally distributed in coastal and oceanic waters (12,29,30). Several members of the genus Dinophysis produce potent polyether toxins that can accumulate in filter-feeding bivalves, leading to a syndrome known as diarrhetic shellfish poisoning (DSP) in humans who consume tainted shellfish. These toxic algal species are important not only for their potential impact on public health but also from an ecological point of view because of their dual role as primary and secondary producers in complex microbial food webs. Despite extensive studies over the last 2 decades, little is known about the ecophysiology, toxicology, and bloom mechanisms of DSP-causing species of Dinophysis, primarily due to an inability to culture them. Since the first successful cultivation of Dinophysis acuminata by Park et al. (42), the understanding of Dinophysis biology and ecology has progressed considerably. Three other species (Dinophysis caudata, Dinophysis fortii, and Dinophysis infundibulus) are now available in culture, and it has become clear that these different Dinophysis species require the presence of both cryptophytes and the marine ciliate Myrionecta rubra to grow and proliferate (36,37,38). When presented with the marine ciliate M. rubra as prey, the four Dinophysis species mentioned...

Kleptoplastidy is the retention of plastids obtained from ingested algal prey, which may remain temporarily functional and be used for photosynthesis by the predator. We showed that the marine dinoflagellate Dinophysis mitra has great kleptoplastid diversity. 8%) were not closely clustered with any particular group. Only six sequences were identical to those of Chrysochromulina simplex, Chrysochromulina hirta, Chrysochromulina sp. TKB8936, Micromonas pusilla NEPCC29, Micromonas pusilla CCMP491, and an unidentified diatom. Thus, we detected >100 different plastid sequences from 14 D. mitra cells, strongly suggesting kleptoplastidy and the need for mixotrophic prey such as Laboea, Tontonia, and Strombidium-like ciliates, which retain numerous symbiotic plastids from different origins, for propagation and plastid sequestration.