New stage of the study on domoic acid-producing diatoms : A finding of Nitzschia navis-varingica that produces domoic acid derivatives as major toxin components

We reported Nitzschia navis-varingica as a new domoic acid (DA)-producing diatom, and furthermore reported its wide distribution in Asian waters. However the surveys were performed only in limited area of each country. In order to obtain more detailed information, screening of N. navis-varingica was primarily performed in the Philippines. During the survey, we confirmed that all strains of N. navis-varingica isolated from Bulacan, Manila Bay did not produce DA, but did produce isodomoic acids A (IA) and B (IB). The confirmation of IA and IB was done by the analyses of UV-spectra, LC-MS/MS, proton NMR and ^C NMR. Re-observation of the toxin composition including IA and IB simultaneously with DA was performed on the former chromatograms. All the strains positive for DA and/or its derivatives isolated from estuary areas of Bacoor, Tanauan and San Roque, were confirmed to produced DA and IB, indicating that there is a local difference in toxin composition in the Philippines. All of the toxic strains were identified as N. navis-varingica. This is the first report of pennate diatom that produces IA and IB instead of DA as major toxin component. This finding might be a useful step in pursuing the DA production mechanism. Additionally fourty one Pseudo-nitzschia strains isolated from offshore areas of above mentioned estuaries were tested for DA, but all of them did not show any sign of DA production

Screening of diatoms producing domoic acid and its derivatives in the Philippines

Domoic acid is the known causative agent responsible for amnesic shellfish poisoning (ASP). Although there is only one documented ASP case in the world, there is a potential of its occurrence in Southeast Asian countries. However, limited information on domoic acid producing diatoms is available except for Nitzschia navis-varingica, which is known to produce significant levels of domoic acid. In order to obtain fundamental data on domoic acid producing diatoms, screening of Pseudo-nitzschia and Nitzschia species were primarily performed in the Philippines. Two source areas, i.e. Manila Bay and Iba estuary of Luzon Island, were selected for observation of these diatoms. Fifty eight isolates of Pseudo-nitzscia and 18 isolates of Nitzschia-like diatoms were prepared from Manila Bay and Iba estuary, respectively. These isolates were cultured and tested for the production of domoic acid and its derivatives. Pseudo-nitzscia strains did not show any signs of domoic acid production. Five out of 18 Nitzschia isolates were confirmed to produce isodomoic acids A and B. Comparison of sonication and boiling in water bath as extraction methods was investigated and results showed that both methods yielded comparable amounts of domoic acid. Stability of domoic acid extracted by boiling was also investigated and was found out to be stable at room temperature for ten days. Results implied an advantageous and convenient way of sample preparation and preservation for international transport

Relationships among Mercury Concentration, and Stable Isotope Ratios of Carbon and Nitrogen in the Scalp Hair of Residents from Seven Countries: Effects of Marine Fish and C4 Plants Consumption.

We analyzed the Hg concentration, and δ¹³C and δ¹⁵N values in the scalp hair of residents from seven countries; Vietnam, New Zealand, Spain, the USA, South Korea, Brazil and Japan. Relationships among the data in each country and among the seven countries were then examined. The highest Hg concentration as well as the highest or higher δ¹⁵N value in each country was found in the hair of a heavy marine fish-eater, whereas the lowest Hg concentration and δ¹⁵N value were found in the hair of a vegetarian or non (marginal)-fish eater. Hg concentrations were positively correlated with the δ¹⁵N values in each country, and increased markedly in samples with δ¹⁵N values exceeding 9.0 ‰, probably due to fish consumption. The highest Hg concentration could be found in sample, with a δ¹³C value between -19 and -18‰, probably reflecting the δ¹³C value of the marine food web

Identification and assessment of domoic acid production in oceanic Pseudo-nitzschia (Bacillariophyceae) from iron-limited waters in the northeast subarctic Pacific

We identified and investigated the potential toxicity of oceanic Pseudo-nitzschia species from Ocean Station Papa (OSP), located in a high-nitrate, low-chlorophyll (HNLC) region of the northeast (NE) subarctic Pacific Ocean. Despite their relatively low abundances in the indigenous phytoplankton assemblage, Pseudo-nitzschia species richness is high. The morphometric characteristics of five oceanic Pseudo-nitzschia isolates from at least four species are described using SEM and TEM. The species identified are Pseudo-nitzschia dolorosa Lundholm et Moestrup, P. granii Hasle, P. heimii Manguin, and P. cf. turgidula (Hust.) Hasle. Additional support for the taxonomic classifications based on frustule morphology is provided through the sequencing of the internal transcribed spacer 1 (ITS1) rDNA. Pseudo-nitzschia species identification was also assessed by the construction of ITS1 clone libraries and using automated ribosomal intergenic spacer analysis (ARISA) for environmental samples collected during the Subarctic Ecosystem Response to Iron Enrichment Study (SERIES), conducted in close proximity to OSP in July of 2002. Based on ITS1 sequences, the presence of P. granii, P. heimii, P. cf. turgidula, and at least five other putative, unidentified Pseudo-nitzschia ITS1 variants was confirmed within iron-enriched phytoplankton assemblages at OSP. None of the oceanic isolates produced detectable levels of particulate domoic acid (DA) when in prolonged stationary phase due to silicic acid starvation. The lack of detectable concentrations of DA suggests that either these strains produce very little or no toxin, or that the physiological conditions required to promote particulate DA production were not met and thus differ from their coastal, toxigenic congeners

Morphological observation of two species of Pseudo-nitzschia (Bacillariophyceae)

About one third of the species in the genus Pseudo-nitzschia has been known to produce domoic acid, which is responsible for amnesic shellfish poisoning, resulting in human intoxication and the death of marine fauna. Precise identification to species level was done based on ultrastructure of frustules marking, which includes fibulae, striae, poroids and central interspace, as well as the cell dimensions. In this study, detail morphological observation of field samples and clonal cultures of Pseudo-nitzschia from estuarine waters in Kuching, Samariang Batu, Santubong and Muara Tebas was carried out using electron microscopy (SEM and TEM). Diatom cells were treated with acid to remove organic cellular contents before observation under SEM and TEM. Two morphotypes were observed from these three locations. One morphotype possesses a rectangular valve shape, the apical and transapical axis of 31.9-36.8μm and 2.6-3.0μm, respectively, with 21-25 fibulae and 21-24 striae in 10μm. The other morphotype shows linear to lanceolate cell shape with the apical axis of 67.2-79.9μm and the transapical axis of 3.0-3.5μm. Numbers of fibulae and striae in 10μm are 12-13 and 11-14, respectively. Central interspace is absent in both morphotypes. Based on the ultrastructure, the two morphotypes of Pseudo-nitzschia is designated as P. brasiliana and P. pungens, respectively.Proceedings of the Horiba International Conference "New Direction of Ocean Research in the Western Pacific" : Past, Present and Future of UNESCO/IOC/WESTPAC Activity for 50 years and the JSPS Project "Coastal Marine Science"Section I: Research Articles/Harmful microalga

Two new ASP toxin production types in strains of Nitzschia navis-varingica from the Philippines

Nitzschia navis-varingica, a diatom widely distributed in brackish waters of Japan and Southeast Asia, was found to produce domoic acid (DA) and its derivatives isodomoic acids A (IA) and B (IB) as the major toxin components. The toxin composition of N. navis-varingica has previously been classified into three types namely DA type (produces DA and IB with an IB ratio of less than 10%), DA-IB type (the IB ratio is 20-80%) and IA-IB type (produces no DA). In the latter type, the ratio of IB tends to be higher in southern areas. In order to investigate more detailed distribution of ASP toxin types of N. navis-varingica, a screening was performed in three localities on Luzon Island, the Philippines. All isolates from one locality, Bulacan, were the DA-IB type. Isolates from the other two localities, Alaminos and Cavite, were found to make up two different and never described toxin composition types, the IB and the DA-IA-IB types, respectively. Sub-strains established from a representative parent of each new toxin composition type showed the same toxin composition as the parental strains. These results suggest that ASP toxin composition types of N. navis-varingica are more complex than previously reported.Proceedings of the Horiba International Conference "New Direction of Ocean Research in the Western Pacific" : Past, Present and Future of UNESCO/IOC/WESTPAC Activity for 50 years and the JSPS Project "Coastal Marine Science"Section I: Research Articles/Harmful microalga