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The consumption of bivalves contaminated with diarrhetic shellfish toxins (DSTs) such as okadaic acid and dinophysistoxins has caused serious economic hardship to shellfish industries worldwide including Japan. Several benthic species of the genus Prorocentrum have been confirmed to produce DSTs. There have been no published studies of Japanese strains of Prorocentrum species that include a three‐way characterization of morphological characteristics, molecular phylogenetic data, and toxin profiles. In the present study, a benthic Prorocentrum strain isolated from Japanese temperate shallow water (depths

Species of the benthic dinoflagellate genus Gambierdiscus are the putative producer of the toxins causing ciguatera fish poisoning (CFP) in tropical and subtropical areas. Although CFP events and distribution of G. caribaeus have been reported in Thailand, little is known about the conditions that enhance the growth of Gambierdiscus in Thailand. Hence, this study was aimed at determining the effects of temperature and salinity and their interactions on the growth of Thai G. caribaeus. The strain tested showed the highest cell yield in IMK/2 medium. Based on growth rate (μ), Thai G. caribaeus grew in the temperature range of 20°C–35°C and the salinity range of 20–40. The suboptimal condition (≥ 80% of the maximal growth rate) for the strain tested was 20.5°C–30.5°C and salinity of 25.0–37.5. The optimal condition was 25°C and salinity of 30. Furthermore, the Thai G. caribaeus revealed high toxicity to mice. Our results suggest that tolerance of up to 35°C may allow these organisms to be distribute...

In Japan, ciguatera fish poisoning (CFP) has been increasingly reported not only in subtropical areas but also in temperate areas in recent years, causing a serious threat to human health. Ciguatera fish poisoning is caused by the consumption of fish that have accumulated toxins produced by an epiphytic/benthic dinoflagellate, genus Gambierdiscus. Previous studies revealed the existence of five Gambierdiscus species/phylotypes in Japan: Gambierdiscus australes, Gambierdiscus scabrosus, Gambierdiscus sp. type 2, Gambierdiscus sp. type 3, and Gambierdiscus (Fukuyoa) cf. yasumotoi. Among these, G. australes, G. scabrosus, and Gambierdiscus sp. type 3 strains exhibited toxicities in mice, whereas Gambierdiscus sp. type 2 strains did not show any toxicity. Therefore, it is important to monitor the cell abundance and dynamics of these species/phylotypes to identify and characterize CFP outbreaks in Japan. Because it is difficult to differentiate these species/phylotypes by observation under a light microscope, development of a rapid and reliable detection and enumeration method is needed. In this study, a quantitative PCR assay was developed using a TaqMan probe that targets unique SSU rDNA sequences of four Japanese Gambierdiscus species/phylotypes and incorporates normalization with DNA recovery efficiency. First, we constructed standard curves with high linearity (R2=1.00) and high amplification efficiency (≥1.98) using linearized plasmids that contained SSU rDNA of the target species/phylotypes. The detection limits for all primer and probe sets were approximately 10 gene copies. Further, the mean number of SSU rDNA copies per cell of each species/phylotype was determined from single cells in culture and from those in environmental samples using the qPCR assay. Next, the number of cells of each species/phylotype in the mixed samples, which were spiked with cultured cells of the four species/phylotypes, was calculated by division of the total number of rDNA copies of each species/phylotype in each sample by the number of rDNA copies per cell. The numbers of cells of each species/phylotype quantified by qPCR assay were similar to the number of cells of each species/phylotype that were spiked. Finally, the cell densities of the target species/phylotypes were quantified using the qPCR assay in 30 environmental samples collected from Japanese coastal areas. Total cell densities of the four Gambierdiscus species/phylotypes quantified by qPCR assay were similar to those of Gambierdiscus spp. quantified by direct counting under a light microscope. The qPCR assay developed in this study is expected to be a powerful new tool for determining detailed distribution patterns and for monitoring the cell abundance and dynamics of each Japanese Gambierdiscus species/phylotype in the coastal areas of Japan.

The toxic benthic dinoflagellate genus Ostreopsis has been connected to the production of palytoxin and its analogs in many tropical and temperate areas. Although the type species, O. siamensis, was originally described from the Gulf of Thailand in 1901, little is known about the species composition and distribution of the genus Ostreopsis in Thailand. In this study, a total of 64 Ostreopsis strains isolated from the Andaman Sea as well as the Gulf of Thailand were investigated by analyzing the nucleotide sequences of the LSU rDNA D1/D2, D8/D10 and ITS-5.8S rDNA regions. Phylogenetic analyses (BI and ML) resulted in some of the strains being assigned to previously described clades, O. cf. ovata and Ostreopsis sp. 6, and revealed the existence of a novel clade named Ostreopsis sp. 7, which exhibited large genetic distances from the other clades. Among O. cf. ovata, several strains from Thailand were formed into a new subclade, the Thailand subclade, whereas a few strains belonged to the South China Sea subclade. Morphometric characteristics such as the cell sizes of the two O. cf. ovata subclades and those of Ostreopsis sp. 7 were not significantly different from each other (p > 0.05). Their characteristics were similar but slightly different from those of O. ovata and were significantly different from those of Ostreopsis sp. 6 (p < 0.05). Toxicities of Ostreopsis from Thailand were evaluated using mouse bioassay. Strains of Ostreopsis sp. 6 and Ostreopsis sp. 7 tested were highly toxic, while the two subclades of O. cf. ovata strains seemed to be nontoxic. This study suggests that toxic Ostreopsis sp. 7 is distributed in the Andaman Sea, whereas the two subclades of O. cf. ovata and toxic Ostreopsis sp. 6 are distributed in the Gulf of Thailand.

A new epiphytic dinoflagellate is described, G ambierdiscus scabrosus sp. nov., from tidal pools and rocky shores along the coastal areas of Japan. Cells are 63.2 ± 5.7 μm in depth, 58.2 ± 5.7 μm in width, and 37.3 ± 3.5 μm in length. The plate formula of G . scabrosus is Po, 4', 0a, 6'', 6c, ?s, 5''', 0p, and 2''''. Morphologically, G . scabrosus resembles G . belizeanus as follows: anterioposteriorly compressed cell shape, narrow 2'''' plate, and areolated surface. Despite this similarity, the cells of G . scabrosus can be distinguishable by the presence of the asymmetric shaped 3'' plate and the rectangular shaped 2' plate.

Monitoring of harmful algal bloom (HAB) species in coastal waters is important for assessment of environmental impacts associated with HABs. Co-occurrence of multiple cryptic species such as toxic dinoflagellate Ostreopsis species make reliable microscopic identification difficult, so the employment of molecular tools is often necessary. Here we developed new qPCR method by which cells of cryptic species can be enumerated based on actual gene number of target species. The qPCR assay targets the LSU rDNA of Ostreopsis spp. from Japan. First, we constructed standard curves with a linearized plasmid containing the target rDNA. We then determined the number of rDNA copies per cell of target species from a single cell isolated from environmental samples using the qPCR assay. Differences in the DNA recovery efficiency was calculated by adding exogenous plasmid to a portion of the sample lysate before and after DNA extraction followed by qPCR. Then, the number of cells of each species was calculated by division of the total number of rDNA copies of each species in the samples by the number of rDNA copies per cell. To test our procedure, we determined the total number of rDNA copies using environmental samples containing no target cells but spiked with cultured cells of several species of Ostreopsis. The numbers estimated by the qPCR method closely approximated total numbers of cells added. Finally, the numbers of cells of target species in environmental samples containing cryptic species were enumerated by the qPCR method and the total numbers also closely approximated the microscopy cell counts. We developed a qPCR method that provides accurate enumeration of each cryptic species in environments. This method is expected to be a powerful tool for monitoring the various HAB species that occur as cryptic species in coastal waters.

BackgroundThe marine epiphytic dinoflagellate genus Gambierdiscus produce toxins that cause ciguatera fish poisoning (CFP): one of the most significant seafood-borne illnesses associated with fish consumption worldwide. So far, occurrences of CFP incidents in Japan have been mainly reported in subtropical areas. A previous phylogeographic study of Japanese Gambierdiscus revealed the existence of two distinct phylotypes: Gambierdiscus sp. type 1 from subtropical and Gambierdiscus sp. type 2 from temperate areas. However, details of the genetic diversity and distribution for Japanese Gambierdiscus are still unclear, because a comprehensive investigation has not been conducted yet.Methods/principal findingA total of 248 strains were examined from samples mainly collected from western and southern coastal areas of Japan during 2006-2011. The SSU rDNA, the LSU rDNA D8-D10 and the ITS region were selected as genetic markers and phylogenetic analyses were conducted. The genetic diversity of Japanese Gambierdiscus was high since five species/phylotypes were detected: including two reported phylotypes (Gambierdiscus sp. type 1 and Gambierdiscus sp. type 2), two species of Gambierdiscus (G. australes and G. cf. yasumotoi) and a hitherto unreported phylotype Gambierdiscus sp. type 3. The distributions of type 3 and G. cf. yasumotoi were restricted to the temperate and the subtropical area, respectively. On the other hand, type 1, type 2 and G. australes occurred from the subtropical to the temperate area, with a tendency that type 1 and G. australes were dominant in the subtropical area, whereas type 2 was dominant in the temperate area. By using mouse bioassay, type 1, type 3 and G. australes exhibited mouse toxicities.Conclusions/significanceThis study revealed a surprising diversity of Japanese Gambierdiscus and the distribution of five species/phylotypes displayed clear geographical patterns in Japanese coastal areas. The SSU rDNA and the LSU rDNA D8-D10 as genetic markers are recommended for further use.

BACKGROUND: A dinoflagellate genus Ostreopsis is known as a potential producer of Palytoxin derivatives. Palytoxin is the most potent non-proteinaceous compound reported so far. There has been a growing number of reports on palytoxin-like poisonings in southern areas of Japan; however, the distribution of Ostreopsis has not been investigated so far. Morphological plasticity of Ostreopsis makes reliable microscopic identification difficult so the employment of molecular tools was desirable. METHODS/PRINCIPAL FINDING: In total 223 clones were examined from samples mainly collected from southern areas of Japan. The D8-D10 region of the nuclear large subunit rDNA (D8-D10) was selected as a genetic marker and phylogenetic analyses were conducted. Although most of the clones were unable to be identified, there potentially 8 putative species established during this study. Among them, Ostreopsis sp. 1-5 did not belong to any known clade, and each of them formed its own clade. The dominant species was Ostreopsis sp. 1, which accounted for more than half of the clones and which was highly toxic and only distributed along the Japanese coast. Comparisons between the D8-D10 and the Internal Transcribed Spacer (ITS) region of the nuclear rDNA, which has widely been used for phylogenetic/phylogeographic studies in Ostreopsis, revealed that the D8-D10 was less variable than the ITS, making consistent and reliable phylogenetic reconstruction possible. CONCLUSIONS/SIGNIFICANCE: This study unveiled a surprisingly diverse and widespread distribution of Japanese Ostreopsis. Further study will be required to better understand the phylogeography of the genus. Our results posed the urgent need for the development of the early detection/warning systems for Ostreopsis, particularly for the widely distributed and strongly toxic Ostreopsis sp. 1. The D8-D10 marker will be suitable for these purposes.