HOT TOPIC 8:
Update of the planktonic diatom genus Pseudo-nitzschia in Aotearoa New Zealand coastal waters: genetic diversity and toxin production.
Tomohiro Nishimura, J. Sam Murray, Michael J. Boundy, Muharrem Balci, Holly A. Bowers, Kirsty F. Smith, D. Tim Harwood, Lesley L. Rhodes.
Toxins 13(9): 637 (2021)
Abstract
Domoic acid (DA) is produced by almost half of the species belonging to the diatom genus Pseudo-nitzschia and causes amnesic shellfish poisoning (ASP). It is, therefore, important to investigate the diversity and toxin production of Pseudo-nitzschia species for ASP risk assessments. Between 2018 and 2020, seawater samples were collected from various sites around Aotearoa New Zealand, and 130 clonal isolates of Pseudo-nitzschia were established. Molecular phylogenetic analysis of partial large subunit ribosomal DNA and/or internal transcribed spacer regions revealed that the isolates were divided into 14 species (Pseudo-nitzschia americana, Pseudo-nitzschia arenysensis, Pseudo-nitzschia australis, Pseudo-nitzschia calliantha, Pseudo-nitzschia cuspidata, Pseudo-nitzschia delicatissima, Pseudo-nitzschia fraudulenta, Pseudo-nitzschia galaxiae, Pseudo-nitzschia hasleana, Pseudo-nitzschia multiseries, Pseudo-nitzschia multistriata, Pseudo-nitzschia plurisecta, Pseudo-nitzschia pungens, and Pseudo-nitzschia cf. subpacifica). The P. delicatissima and P. hasleana strains were further divided into two clades/subclades (I and II). Liquid chromatography-tandem mass spectrometry was used to assess the production of DA and DA isomers by 73 representative strains. The analyses revealed that two (P. australis and P. multiseries) of the 14 species produced DA as a primary analogue, along with several DA isomers. This study is the first geographical distribution record of P. arenysensis, P.cuspidata, P. galaxiae, and P. hasleana in New Zealand coastal waters.


HOT TOPIC 7:
First report of Alexandrium (Dinophyceae) associated with marine macroalgae off Japan: Diversity, distribution, and toxicity.
Tomohiro Nishimura, Yuki Kuribara, Ryo Fukuzawa, Katsuya Mimura, Hiroshi Funaki, Kouki Tanaka, Ryuichi Watanabe, Hajime Uchida, Toshiyuki Suzuki, Masao Adachi.
Harmful Algae 104: 101924 (2021)
Abstract
Macroalgal samples were collected from coastal waters in subboreal to subtropical zones in Japan (< 3–30 m depths) and 32 clonal strains of non-motile dinoflagellate-like protists were established. Molecular phylogenetic analyses of the LSU rDNA D1/D2, SSU rDNA, ITS region, and concatenated SSU rDNA + LSU rDNA D1/D2 sequences revealed that the strains nested within the genus Alexandrium. They were separated into three novel phylotypes: Alexandrium spp. type 1, type 2, and type 3. Analysis of the concatenated sequences revealed that the most closely related species for the three phylotypes was A. ostenfeldii. Most cells from strains of the three phylotypes were non-motile and hemispherical to spherical in shape. The average diameters of the non-motile cells were between 35 and 39 µm. Type 1 and type 2 were widely distributed in Japan from the temperate to subtropical zones, whereas type 3 was restricted to the temperate zone. Furthermore, type 2 was widespread from shallow to deep waters, whereas type 1 and type 3 were restricted to deep waters. Growth experiments in strains belonging to the three phylotypes revealed that the occurrence ratios of motile cells were very low (≤ 1.1% of the total cells). The production of paralytic shellfish poisoning toxins, tetrodotoxin, and cyclic imines was assessed in strains belonging to the three phylotypes by LC/MS/MS analysis. The strains did not produce any of the toxins tested. The strains of the three phylotypes showed lethal toxicity to mice by intraperitoneal administration. To the best of our knowledge, this is the first study to report the existence of Alexandrium associated with marine macroalgae from Japan.


HOT TOPIC 6:
Abundance of the benthic dinoflagellate Prorocentrum and the diversity, distribution, and diarrhetic shellfish toxin production of Prorocentrum lima complex and P. caipirignum in Japan.
Tomohiro Nishimura, Hajime Uchida, Ryoko Noguchi, Hiroshi Oikawa, Toshiyuki Suzuki, Hiroshi Funaki, Chiho Ihara, Kyoko Hagino, Shingo Arimitsu, Yuta Tanii, Shota Abe, Kana Hashimoto, Katsuya Mimura, Kouki Tanaka, Ippei Yanagida, Masao Adachi.
Harmful Algae 96: 101687 (2020) 
Abstract
In the present study, the abundance of Prorocentrum and the molecular phylogeny, distribution, and DST production of P. lima complex and P. caipirignum in Japan were investigated. First, the cell densities of Prorocentrum were assessed from the temperate to subtropical zones in Japan between 2014 and 2018. The cell density in the subtropical zone [19.0 ± 40.2 cells/g wet weight (ww) algae] was significantly higher than that in the temperate zone (1.4 ± 3.4 cells/g ww algae). A total of 244 clonal strains were established from the temperate and subtropical zones. Phylogenetic analyses based on the large-subunit ribosomal DNA D1/D2 revealed that the strains were separated into four species/species complex/phylotypes (P. lima complex, P. caipirignum, and new phylotypes Prorocentrum spp. types 1 and 2). The strains of P. lima complex could be separated into two clades (1 and 3). Furthermore, the strains of clades 1 and 3 could be separated into nine subclades (1a, 1c, 1d, 1e, 1f, 1g, 1h, 1i, and 1j) and three subclades (3a, 3b, and 3c), respectively. The strains of P. caipirignum were separated into two subclades (b and e). Each phylotype/subclade showed a unique distribution pattern in Japan: P. lima complex subclades 1a, 1c, and 3a and P. caipirignum subclades b and e were widespread from the temperate to subtropical zones. On the other hand, P. lima complex subclades 1e and 1i were restricted to the temperate zone, and P. lima complex subclades 1d, 1f, 1 g, 1 h, 1 j, 3b, and 3c and Prorocentrum spp. types 1 and 2 were restricted to the subtropical zone. Furthermore, the DST production of the 243 clonal strains was assessed by LC/MS/MS analysis. The results revealed that all strains produced okadaic acid (OA) and that the OA contents of P. lima complex subclades 1d and 1f, P. caipirignum subclades b and e, and Prorocentrum sp. type 2 tended to be higher than those of the other subclades. While P. lima complex subclades 1a, 1e, 1f, and 1i produced DTX1, the other phylotype/subclades produced either no or low quantities of DTX1. A strain of P. lima complex subclade 1e showed the highest OA and DTX1 contents (55.27 and 70.73 pg/cell, respectively) in the world. These results suggest that there are potential risks for DST accumulation in benthic animals in Japan.


HOT TOPIC 5:
First report on okadaic acid production of a benthic dinoflagellate Prorocentrum cf. fukuyoi from Japan.
Tomohiro Nishimura, Hajime Uchida, Toshiyuki Suzuki, Wittaya Tawong, Shota Abe, Shingo Arimitsu, Masao Adachi.
Phycological Research 68(1): 30-40 (2020) 
Abstract
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 < 3 m) was characterized using morphological, phylogenetic, and toxicological methods. Cell size of the Japanese Prorocentrum strain KSK4P was 27.2 ± 1.3 μm in length, 22.4 ± 0.9 μm in width, and had a length-to-width ratio of 1.22 ± 0.04. The thecal surface was smooth and covered with large and small thecal pores lying in rows, and the pores were lacking in the central area of the theca. The right theca had a deeply excavated periflagellar area that was narrow V-shaped. Nine platelets and two wings were in the periflagellar area. These morphological features of the strain KSK4P are similar to those of P. fukuyoi. Phylogenetic trees based on the LSU rDNA D1/D3 sequences revealed that the strain KSK4P forms a new subclade F2f belonging to P. fukuyoi complex (subclade F2). The sequence analysis revealed that the strain KSK4P (subclade F2f) diverges from a subclade F2e to which the strain used for the description of P. fukuyoi belongs. Based on the results of morphological and phylogenetic analyses of the strain KSK4P, the strain was designated as P. cf. fukuyoi. The results of the LC/MS/MS analysis revealed that the strain KSK4P produced okadaic acid (1.5-2.7 fg cell−1). This is the first report that a strain belonging to the P. fukuyoi complex can produce okadaic acid.


HOT TOPIC 4:
Abundance and seasonal population dynamics of the potentially ciguatera-causing dinoflagellate Gambierdiscus in Japanese coastal areas between 2007 and 2013.
Tomohiro Nishimura, Wittaya Tawong, Hiroshi Sakanari, Takuji Ikegami, Keita Uehara, Daiki Inokuchi, Masatoshi Nakamura, Takuya Yoshioka, Shota Abe, Haruo Yamaguchi, Masao Adachi.
Plankton and Benthos Research 13(2): 46-58 (2018) 
Abstract
Ciguatera fish poisoning (CFP) is caused by toxins originating from an epiphytic/benthic dinoflagellate of the genus Gambierdiscus. In Japan, CFP cases have been increasingly reported not only in subtropical areas but also in temperate areas. It is therefore important to study Gambierdiscus cell occurrences, cell densities, and population dynamics to address CFP outbreaks in Japan. This study assessed the densities in Japanese shallow waters (0.1–3 m depths) and revealed that the densities were lower than those in tropical and subtropical areas worldwide. In the shallow waters of Tosa Bay, a Japanese temperate area, population dynamics of Gambierdiscus cells were assessed monthly between 2007 and 2013. Gambierdiscus did not show substrate preferences for macroalgal species. The cell densities in the area ranged from 0 to 232.2 cells g−1 wet weight algae. The average cell densities in spring, summer, autumn, and winter were 0.1±0.4, 0.9±2.6, 4.0±20.6, and 0.4±1.4 cells g−1 wet weight algae, respectively. The cell densities in summer and autumn were not significantly different (p>0.05), whereas those in summer and autumn were significantly higher than those in spring and winter (p<0.01). A significant positive correlation between cell densities and sea surface temperatures (SSTs) was observed (rs=0.21, p<0.001), while a significant negative correlation between cell densities and salinity was recognized (rs=−0.18, p<0.001). These results suggest that cell densities of Gambierdiscus in Japanese temperate shallow waters increase in summer and autumn when the SST is high and salinity is moderately low.


HOT TOPIC 3:
Quantitative PCR assay for detection and enumeration of ciguatera-causing dinoflagellate Gambierdiscus spp. (Gonyaulacales) in coastal areas of Japan.
Tomohiro Nishimura, Naohito Hariganeya, Wittaya Tawong, Hiroshi Sakanari, Haruo Yamaguchi, Masao Adachi.
Harmful Algae 52: 11-22 (2016) 
Abstract
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.



HOT TOPIC 2:
Morphology of Gambierdiscus scabrosus sp. nov. (Gonyaulacales): a new epiphytic toxic dinoflagellate from coastal areas of Japan 
Tomohiro Nishimura, Shinya Sato, Wittaya Tawong, Hiroshi Sakanari, Haruo Yamaguchi and Masao Adachi
Journal of Phycology 50(3): 506-514 (2014)
Abstract
A new epiphytic dinoflagellate is described, Gambierdiscus 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.



HOT TOPIC 1:
Genetic diversity and distribution of the ⅽiguatera-ⅽausing dinoflagellate Gambierdiscus spp. (Dinophyceae) in ⅽoastal areas of Japan
Tomohiro Nishimura, Shinya Sato, Wittaya Tawong, Hiroshi Sakanari, Keita Uehara, Md Mahfuzur Rahman Shah, Shoichiro Suda, Takeshi Yasumoto,Yohsuke Taira, Haruo Yamaguchi, Masao Adachi
PLOS ONE 8: e60882 (2013)
Abstract
Background: The 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 Finding: A 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/Significance: This 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.