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Confirmation of domoic acid production of Pseudo-nitzschia multiseries isolated from Ofunato Bay, Japan
PERGAMON
Toxicon 37 (1999) 677±682
Short communication
Con®rmation of domoic acid production of Pseudo-nitzschia multiseries isolated from Ofunato Bay, Japan Yuichi Kotaki a, Kazuhiko Koike a, Shigeru Sato a, Takehiko Ogata a, Yasuwo Fukuyo b, Masaaki Kodama a, * a School of Fisheries Sciences, Kitasato University, Sanriku, Iwate 022-0101, Japan Asian Natural Environmental Science Center, The University of Tokyo, Tokyo 113-0032, Japan
b
Received 3 July 1998; accepted 8 September 1998
Abstract Production of domoic acid (DA), the responsible toxin for amnesic shell®sh poisoning, was examined for 44 strains of Pseudo-nitzschia spp. isolated from Ofunato Bay, Japan. Only one strain which was identi®ed as Pseudo-nitzschia multiseries produced DA in a level comparable to Canadian strains. No signi®cant DA was detected in the rest of the strains, indicating that toxic P. multiseries does not bloom in a high density in the bay. # 1999 Elsevier Science Ltd. All rights reserved.
Amnesic shell®sh poisoning (ASP) is a new type of shell®sh poisoning, the occurrence of which was ®rst recognized in Prince Edward Island, Canada, in 1987 (Addison and Stewart, 1989; Perl et al., 1990). The neurotoxin responsible for the poisoning has been identi®ed as domoic acid (DA) (Wright et al., 1989), a neuroexcitatory amino acid related to glutamate, which had been isolated from marine red algae belonging to the order Ceramiales (Takemoto and Daigo, 1958). In the incidence in Canada, this substance was detected in the diatom Pseudonitzschia multiseries (former Nitzschia pungens f. multiseries) as well as in the shell®sh, and thus this species is identi®ed as a causative organism of the poisoning (Bates et al., 1989). Since the incidence in Canada, DA productivity of * Corresponding author. Tel.: +81-192-44-2121; fax: +81-192-44-2125; e-mail: m-kodama@ nnet.ne.jp 0041-0101/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 1 - 0 1 0 1 ( 9 8 ) 0 0 2 1 0 - 4
678
Y. Kotaki et al. / Toxicon 37 (1999) 677±682
P. multiseries has been reported from other parts of the world (e.g. Vrieling et al., 1996). Other species of Pseudo-nitzschia such as P. australis (Garrison et al., 1992) and P. pseudodelicatissima (Martin et al., 1990) were also found to be toxic. These ®ndings imply that Pseudo-nitzschia spp. potentially involved in ASP distribute widely in the world. On the other hand, no survey on ASP has been conducted in Japanese coastal waters, though P. multiseries was listed as one of the members of the phytoplankton community there (Takano and Kuroki, 1977). We report here with clear chemical evidence that P. multiseries isolated from Ofunato Bay, Japan, produces a high level of DA in culture. Water and vertical net hauling samples (20 mm) were collected at Shizu station (24 m depth) in Ofunato Bay (Ogata et al., 1982) every week from April to September 1994, and every month from October 1994 to February 1995. Five specimens of the indicator bivalves (mussel Mytilus edudis and scallop Patinopecten yessoensis) set at the station at around 2 m (mussel) and 10 m (scallop) depths were collected monthly from April 1994 to February 1995. Each single cell of Pseudo-nitzschia spp. found in the net hauling or water samples was inoculated to f/2 medium (Guillard and Ryther, 1962) in Nunc's multihole plates and cultured at 158C under light intensity of 100 mmol photon mÿ2 sÿ1 with 16:8 LD cycle. Strains succeeded in culture were kept in 30 ml of the medium under the same conditions. Two weeks after the culture had reached the stationary phase at which the DA production of P. multiseries was reported to increase (Bates et al., 1991), DA level in the culture was analyzed by HPLC with pre-column derivatization according to Pocklington et al. (1990). In total, 44 strains of Pseudo-nitzschia spp. were cultured; 1 strain in June, 24 in October, 10 in November and 9 in December. Among these strains, one strain (OFP941) collected in October showed a big and clear peak at the identical retention time to that of standard DA, whereas no signi®cant DA-like peak was observed in the extracts of the other strains. Therefore, the substance which gave a peak at the identical retention time to DA was puri®ed from a large scale culture (3 l) of the strain by Amberlite XAD-2 and ODS-5 column chromatographies according to Wright et al. (1989). The ®nal yield of the substance was 370 mg from the cells and 800 mg from the medium, respectively. ESI/MS of both substances showed ion peaks at m/z = 312 and 334 which correspond with (M + H)+ and (M + Na)+ of DA, indicating that the substances in both samples are identical. For further con®rmation, these samples were combined and analyzed by proton NMR. Fig. 1 shows the proton NMR spectrum which is consistent with that of authentic DA (Diagnostic Chemicals, Canada) analyzed under the same conditions. These results show that the substance is DA. The morphological characteristics of the DA-producing strain observed under a transmission electron microscope are fully accordant with the description of P. multiseries by Hasle (1995). Pseudonodule is absent; frustule has 13±15 keel puncta and 13±19 transapical costae in 10 mm; intercostal membrane is perforated by three or four rows of closely spaced poroids which have 5±7 pores in 1 mm; the length is 76.4±102.5 mm, and the width 2.9±4.0 mm.
Y. Kotaki et al. / Toxicon 37 (1999) 677±682
679
Fig. 1. Proton NMR spectrum of the DA-like substance puri®ed from the Ofunato strain of Pseudonitzschia multiseries. The spectrum was measured in 4% CD3COOD/D2O on a Varian 500 MHz instrument. The signal due to CHD2COOD at 2.06 ppm was used as reference.
The DA production of P. multiseries is reported to increase at the late stationary phase of the batch culture (Bates et al., 1991). Thus the DA productivity of the strain (OFP941) was analyzed under various growth stages in the culture. As shown in Fig. 2, the cells grew logarithmically during the ®rst 6 days with a division rate of 0.92 times dayÿ1, reaching a maximum density of 159,000 cells mlÿ1 at day 12, and then decreased gradually to 60,000 cells mlÿ1 over a further 15 days. The initial DA level of the cells was 3.1 pg cellÿ1. This decreased to less than 0.2 pg cellÿ1 within 6 days and did not increase signi®cantly during the exponential and early stationary phase of the growth. At day 15 when the cell number was decreasing because of cell lysis, the DA level of the cells started to increase markedly to 5.7 pg cellÿ1 (day 21) which is comparable to the toxicity of strongly toxic strains in Canada (Bates et al., 1989). The increasing rate then slightly slowed down during day 21 and 27. During this period, considerable amounts of DA were detected in the cell-free medium whereas no signi®cant DA was detected in the medium until day 21, indicating that DA is released from the cells to the medium in this period. It is reported by several authors that P. multiseries in a batch culture produces DA when the cell growth is in the late stationary phase (Bates et al., 1991; Osada and Stewart, 1997). This pattern in DA production is also reported in P. australis (Garrison et al., 1992). The Ofunato strain of P. multiseries showed similar characteristics for DA production in a batch culture (Fig. 2). The level of DA in the cells just after the inoculation, which seems to be due to carryover from the stationary phase, decreased within 6 days,
680
Y. Kotaki et al. / Toxicon 37 (1999) 677±682
Fig. 2. Cell growth and DA production of Pseudo-nitzschia multiseries. P. multiseries strain acclimatized to the conditions was inoculated into 2 l of f/2 medium (Guillard and Ryther, 1962) with the initial cell concentration of 1,700 cells mlÿ1 and cultured for 27 days at 158C under light intensity of 100 mmol photon mÿ2 sÿ1 with 16:8 LD cycle. DA levels of the cell and culture medium were monitored separately by the HPLC-¯uorescent analysis according to Pocklington et al. (1990). The growth was monitored by counting cell number under light microscope.
when the cells began to grow logarithmically (Douglas and Bates, 1992). These data support the fact that P. multiseries does not produce DA when there is an excess amount of nutrients in the environment (Bates et al., 1991; Osada and Stewart, 1997). In the analysis by HPLC (Quilliam et al., 1989) on the 50% MeOH extracts prepared from the scallop digestive gland and the whole tissue of mussel after treatment by Sep-Pak C18 cartridge, a peak corresponding with DA was observed in bivalves collected during November 1994 and February 1995. However, the highest toxicity was 2.8 and 0.8 mg gÿ1 for mussel and scallop, respectively, which was much lower than the quarantine level for safety consumption in Canada (20 mg gÿ1) (Iverson and Truelove, 1994). Since the ®rst incidence in Canada, DA-producing Pseudo-nitzschia spp. have been reported from various parts of the world (Villac et al., 1993). The results of the present study showed with clear chemical evidence that one strain of P. multiseries isolated from Ofunato Bay produces DA with a productivity comparable to Canadian strains (Bates et al., 1991). Takano and Kuroki (1977) reported that P. multiseries is one of the members of the phytoplankton community in Ofunato Bay. This indicates that P. multiseries has not been recently introduced, but has been well adopted to survival in this area. However, no ASP or ASP-like poisoning has been recognized there. These suggest that toxic P. multiseries does not bloom in a high density in the bay. This is supported by the present results that no signi®cant DA contamination was observed in the shell®sh in the bay.
Y. Kotaki et al. / Toxicon 37 (1999) 677±682
681
Because of the diculty in identifying toxic species belonging to the genus Pseudo-nitzschia under light microscopy, the screening of toxic species was carried out by analyzing the DA production of as many isolates as possible. In total, 44 strains of Pseudo-nitzschia spp. were cultured during June and December among which only one strain was found to be toxic. This also supports the argument that toxic P. multiseries does not bloom in a high density in Ofunato Bay. In the case of the causative dino¯agellates of paralytic shell®sh poisoning, nontoxic strains are often observed in toxic species (Yentsch et al., 1978). Similar ®ndings are observed in the DA-producing species. Amphora coaeiformis isolated from Seal River, o Cardigan Bay, Prince Edward Island, is reported to produce DA (Maranda et al., 1990). In contrast, DA was not detected in the strain of the same species from culture collections in the Provasoli-Guillard Center for Marine Phytoplankton (Bates et al., 1989). In the present study, the species identi®cation was carried out only on the strain which produced DA in the culture conditions described above. It is so far not clear whether P. multiseries is included in the rest of the 43 strains, though there is no report on the nontoxic strains of P. multiseries. Species identi®cation of nontoxic strains is under progress.
Acknowledgements We express our sincere thank to Dr. T. Kawamura, Tohoku National Fisheries Research Institute, for his helpful advice in species identi®cation. We also thank Dr. R. Sakai of Kitasato University and Dr. M. Satake of Tohoku University for taking NMR and ESI/MS. This work was partly supported by a grant from the Fisheries Agency of Japan.
References Addison, R.F., Stewart, J.E., 1989. Domoic acid and the eastern Canadian molluscan shell®sh industry. Aquaculture 77, 263±269. Bates, S.S., Bird, C.J., De Freitas, A.S.W., Foxall, R., Gilgan, M., Hanic, L.A., Jhonson, G.R., McCulloch, A.W., Odense, P., Pocklington, R., Quilliam, M.A., Sim, P.G., Smith, J.C., Rao, D.V.S., Todd, E.C.D., Walter, J.A., Wright, J.L.C., 1989. Pennate diatom Nitzschia pungens as the primary source of domoic acid, a toxin in shell®sh from eastern Prince Edward Island, Canada. Can. J. Fish. Aquat. Sci. 46, 1203±1215. Bates, S.S., De Freitas, A.S.W., Milley, J.E., Pocklington, R., Quilliam, M.A., Smith, J.C., Worms, J., 1991. Controls on domoic acid production by the diatom Nitzschia pungens f. multiseries in culture: Nutrients and irradiance. Can. J. Fish. Aquat. Sci. 48, 1136±1144. Douglas, D.J., Bates, S.S., 1992. Production of domoic acid, a neurotoxic amino acid, by an axenic culture of the marine diatom Nitzschia pungens f. multiseries Hasle. Can. J. Fish. Aquat. Sci. 49, 85±90. Garrison, D.L., Conrad, S.M., Eilers, P.P., Waldron, E.M., 1992. Con®rmation of domoic acid production by Pseudonitzschia australis (Bacillariophyceae) cultures. J. Phycol. 28, 604±607. Guillard, R.R.L., Ryther, J.H., 1962. Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt, and Detonula confervacea (Cleve) Gran. Can. J. Microbiol. 8, 229±239.
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Hasle, G.R., 1995. Pseudo-nitzschia pungens and P. multiseries (Bacillariophyceae): Nomenclatural history, morphology, and distribution. J. Phycol. 31, 428±435. Iverson, F., Truelove, J., 1994. Toxicology and seafood toxins: Domoic acid. Natural Toxins 2, 334±339. Maranda, L., Wang, R., Masuda, K., Shimizu, Y., 1990. Investigation of the source of domoic acid in mussels. In: Graneli, E., Sundstroem, B., Edler, L., Anderson, D.M. (Eds.), Toxic Marine Phytoplankton. Elsevier Science Publishing Co., New York, pp. 300±304. Martin, J.L., Haya, K., Burridge, L.E., Wildish, D.J., 1990. Nitzschia pseudodelicatissima ± a source of domoic acid in the Bay of Fundy, eastern Canada. Mar. Ecol. Prog. Ser. 67, 177±182. Ogata, T., Kodama, M., Fukuyo, Y., Inoue, T., Kamiya, H., Matsuura, F., Sekiguchi, K., Watanabe, S., 1982. The occurrence of Protogonyaulax spp. in Ofunato Bay, in association with the toxi®cation of the scallop Patinopecten yessoensis. Bull. Japan. Soc. Sci. Fish. 48, 563±566. Osada, M., Stewart, J.E., 1997. Gluconic acid/gluconolactone: physiological in¯uences on domoic acid production by bacteria associated with Pseudo-nitzschia multiseries. Aquat. Microb. Ecol. 12, 203±209. Perl, T.M., Bedard, L., Kosatsky, T., Hockin, J.C., Todd, E.C.D., Remis, R.S., 1990. An outbreak of toxic encephalopathy caused by eating mussels contaminated with domoic acid. New Engl. J. Med. 322, 1775±1780. Pocklington, R., Milley, J.E., Bate, S.S., Bird, C.J., De Freitas, A.S.W., Quilliam, M.A., 1990. Trace determination of domoic acid in seawater and phytoplankton by high-performance liquid chromatography of the ¯uorenylmethoxycarbonyl (FMOC) derivative. Int. J. Environ. Anal. Chem. 38, 351±368. Quilliam, M.A., Sim, P.G., McCulloch, A.W., McInnes, A.G., 1989. High-performance liquid chromatography of domoic acid, a marine neurotoxin, with application to shell®sh and plankton. Int. J. Environ. Anal. Chem. 36, 139±154. Takano, H., Kuroki, K., 1977. Some diatoms in the section Pseudonitzschia found in coastal waters of Japan. Bull. Tokai Reg. Fish. Res. Lab. 91, 41±51. Takemoto, T., Daigo, K., 1958. Constituents of Chondria armata. Chem. Pharm. Bull. 6, 578±580. Villac, M.C., Roelke, D.L., Villareal, T.A., Fryxell, G.A., 1993. Comparison of two domoic acid-producing diatoms: a review. Hydrobiologia 269±270, 213±224. Vrieling, E.G., Koeman, R.P.T., Scholin, C.A., Scheerman, P., Peperzak, L., Veenhuis, M., Gieskes, W.W.C., 1996. Identi®cation of a domoic acid-producing Pseudo-nitzschia species (Bacillariophyceae) in the Dutch Wadden Sea with electron microscopy and molecular probes. Eur. J. Phycol. 31, 333±340. Wright, J.L.C., Boyd, R.K., De Freitas, A.S.W., Falk, M., Foxall, R.A., Jamieson, W.D., Laycock, M.V., McCulloch, A.W., McInnes, A.G., Odense, P., Pathak, V.P., Quilliam, M.A., Ragan, M.A., Sim, P.G., Thibault, P., Walter, J.A., Gilgan, M., Richard, D.J.A., Dewar, D., 1989. Identi®cation of domoic acid, a neuroexcitatory amino acid, in toxic mussels from eastern Prince Edward Island. Can. J. Chem. 67, 481±490. Yentsch, C.M., Dale, B., Hurst, J.W., 1978. Coexistence of toxic and nontoxic dino¯agellates resembling Gonyaulax tamarensis in New England coastal waters (NW Atlantic). J. Phycol. 14, 330±332.
Toxicon 37 (1999) 677±682
Short communication
Con®rmation of domoic acid production of Pseudo-nitzschia multiseries isolated from Ofunato Bay, Japan Yuichi Kotaki a, Kazuhiko Koike a, Shigeru Sato a, Takehiko Ogata a, Yasuwo Fukuyo b, Masaaki Kodama a, * a School of Fisheries Sciences, Kitasato University, Sanriku, Iwate 022-0101, Japan Asian Natural Environmental Science Center, The University of Tokyo, Tokyo 113-0032, Japan
b
Received 3 July 1998; accepted 8 September 1998
Abstract Production of domoic acid (DA), the responsible toxin for amnesic shell®sh poisoning, was examined for 44 strains of Pseudo-nitzschia spp. isolated from Ofunato Bay, Japan. Only one strain which was identi®ed as Pseudo-nitzschia multiseries produced DA in a level comparable to Canadian strains. No signi®cant DA was detected in the rest of the strains, indicating that toxic P. multiseries does not bloom in a high density in the bay. # 1999 Elsevier Science Ltd. All rights reserved.
Amnesic shell®sh poisoning (ASP) is a new type of shell®sh poisoning, the occurrence of which was ®rst recognized in Prince Edward Island, Canada, in 1987 (Addison and Stewart, 1989; Perl et al., 1990). The neurotoxin responsible for the poisoning has been identi®ed as domoic acid (DA) (Wright et al., 1989), a neuroexcitatory amino acid related to glutamate, which had been isolated from marine red algae belonging to the order Ceramiales (Takemoto and Daigo, 1958). In the incidence in Canada, this substance was detected in the diatom Pseudonitzschia multiseries (former Nitzschia pungens f. multiseries) as well as in the shell®sh, and thus this species is identi®ed as a causative organism of the poisoning (Bates et al., 1989). Since the incidence in Canada, DA productivity of * Corresponding author. Tel.: +81-192-44-2121; fax: +81-192-44-2125; e-mail: m-kodama@ nnet.ne.jp 0041-0101/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 1 - 0 1 0 1 ( 9 8 ) 0 0 2 1 0 - 4
678
Y. Kotaki et al. / Toxicon 37 (1999) 677±682
P. multiseries has been reported from other parts of the world (e.g. Vrieling et al., 1996). Other species of Pseudo-nitzschia such as P. australis (Garrison et al., 1992) and P. pseudodelicatissima (Martin et al., 1990) were also found to be toxic. These ®ndings imply that Pseudo-nitzschia spp. potentially involved in ASP distribute widely in the world. On the other hand, no survey on ASP has been conducted in Japanese coastal waters, though P. multiseries was listed as one of the members of the phytoplankton community there (Takano and Kuroki, 1977). We report here with clear chemical evidence that P. multiseries isolated from Ofunato Bay, Japan, produces a high level of DA in culture. Water and vertical net hauling samples (20 mm) were collected at Shizu station (24 m depth) in Ofunato Bay (Ogata et al., 1982) every week from April to September 1994, and every month from October 1994 to February 1995. Five specimens of the indicator bivalves (mussel Mytilus edudis and scallop Patinopecten yessoensis) set at the station at around 2 m (mussel) and 10 m (scallop) depths were collected monthly from April 1994 to February 1995. Each single cell of Pseudo-nitzschia spp. found in the net hauling or water samples was inoculated to f/2 medium (Guillard and Ryther, 1962) in Nunc's multihole plates and cultured at 158C under light intensity of 100 mmol photon mÿ2 sÿ1 with 16:8 LD cycle. Strains succeeded in culture were kept in 30 ml of the medium under the same conditions. Two weeks after the culture had reached the stationary phase at which the DA production of P. multiseries was reported to increase (Bates et al., 1991), DA level in the culture was analyzed by HPLC with pre-column derivatization according to Pocklington et al. (1990). In total, 44 strains of Pseudo-nitzschia spp. were cultured; 1 strain in June, 24 in October, 10 in November and 9 in December. Among these strains, one strain (OFP941) collected in October showed a big and clear peak at the identical retention time to that of standard DA, whereas no signi®cant DA-like peak was observed in the extracts of the other strains. Therefore, the substance which gave a peak at the identical retention time to DA was puri®ed from a large scale culture (3 l) of the strain by Amberlite XAD-2 and ODS-5 column chromatographies according to Wright et al. (1989). The ®nal yield of the substance was 370 mg from the cells and 800 mg from the medium, respectively. ESI/MS of both substances showed ion peaks at m/z = 312 and 334 which correspond with (M + H)+ and (M + Na)+ of DA, indicating that the substances in both samples are identical. For further con®rmation, these samples were combined and analyzed by proton NMR. Fig. 1 shows the proton NMR spectrum which is consistent with that of authentic DA (Diagnostic Chemicals, Canada) analyzed under the same conditions. These results show that the substance is DA. The morphological characteristics of the DA-producing strain observed under a transmission electron microscope are fully accordant with the description of P. multiseries by Hasle (1995). Pseudonodule is absent; frustule has 13±15 keel puncta and 13±19 transapical costae in 10 mm; intercostal membrane is perforated by three or four rows of closely spaced poroids which have 5±7 pores in 1 mm; the length is 76.4±102.5 mm, and the width 2.9±4.0 mm.
Y. Kotaki et al. / Toxicon 37 (1999) 677±682
679
Fig. 1. Proton NMR spectrum of the DA-like substance puri®ed from the Ofunato strain of Pseudonitzschia multiseries. The spectrum was measured in 4% CD3COOD/D2O on a Varian 500 MHz instrument. The signal due to CHD2COOD at 2.06 ppm was used as reference.
The DA production of P. multiseries is reported to increase at the late stationary phase of the batch culture (Bates et al., 1991). Thus the DA productivity of the strain (OFP941) was analyzed under various growth stages in the culture. As shown in Fig. 2, the cells grew logarithmically during the ®rst 6 days with a division rate of 0.92 times dayÿ1, reaching a maximum density of 159,000 cells mlÿ1 at day 12, and then decreased gradually to 60,000 cells mlÿ1 over a further 15 days. The initial DA level of the cells was 3.1 pg cellÿ1. This decreased to less than 0.2 pg cellÿ1 within 6 days and did not increase signi®cantly during the exponential and early stationary phase of the growth. At day 15 when the cell number was decreasing because of cell lysis, the DA level of the cells started to increase markedly to 5.7 pg cellÿ1 (day 21) which is comparable to the toxicity of strongly toxic strains in Canada (Bates et al., 1989). The increasing rate then slightly slowed down during day 21 and 27. During this period, considerable amounts of DA were detected in the cell-free medium whereas no signi®cant DA was detected in the medium until day 21, indicating that DA is released from the cells to the medium in this period. It is reported by several authors that P. multiseries in a batch culture produces DA when the cell growth is in the late stationary phase (Bates et al., 1991; Osada and Stewart, 1997). This pattern in DA production is also reported in P. australis (Garrison et al., 1992). The Ofunato strain of P. multiseries showed similar characteristics for DA production in a batch culture (Fig. 2). The level of DA in the cells just after the inoculation, which seems to be due to carryover from the stationary phase, decreased within 6 days,
680
Y. Kotaki et al. / Toxicon 37 (1999) 677±682
Fig. 2. Cell growth and DA production of Pseudo-nitzschia multiseries. P. multiseries strain acclimatized to the conditions was inoculated into 2 l of f/2 medium (Guillard and Ryther, 1962) with the initial cell concentration of 1,700 cells mlÿ1 and cultured for 27 days at 158C under light intensity of 100 mmol photon mÿ2 sÿ1 with 16:8 LD cycle. DA levels of the cell and culture medium were monitored separately by the HPLC-¯uorescent analysis according to Pocklington et al. (1990). The growth was monitored by counting cell number under light microscope.
when the cells began to grow logarithmically (Douglas and Bates, 1992). These data support the fact that P. multiseries does not produce DA when there is an excess amount of nutrients in the environment (Bates et al., 1991; Osada and Stewart, 1997). In the analysis by HPLC (Quilliam et al., 1989) on the 50% MeOH extracts prepared from the scallop digestive gland and the whole tissue of mussel after treatment by Sep-Pak C18 cartridge, a peak corresponding with DA was observed in bivalves collected during November 1994 and February 1995. However, the highest toxicity was 2.8 and 0.8 mg gÿ1 for mussel and scallop, respectively, which was much lower than the quarantine level for safety consumption in Canada (20 mg gÿ1) (Iverson and Truelove, 1994). Since the ®rst incidence in Canada, DA-producing Pseudo-nitzschia spp. have been reported from various parts of the world (Villac et al., 1993). The results of the present study showed with clear chemical evidence that one strain of P. multiseries isolated from Ofunato Bay produces DA with a productivity comparable to Canadian strains (Bates et al., 1991). Takano and Kuroki (1977) reported that P. multiseries is one of the members of the phytoplankton community in Ofunato Bay. This indicates that P. multiseries has not been recently introduced, but has been well adopted to survival in this area. However, no ASP or ASP-like poisoning has been recognized there. These suggest that toxic P. multiseries does not bloom in a high density in the bay. This is supported by the present results that no signi®cant DA contamination was observed in the shell®sh in the bay.
Y. Kotaki et al. / Toxicon 37 (1999) 677±682
681
Because of the diculty in identifying toxic species belonging to the genus Pseudo-nitzschia under light microscopy, the screening of toxic species was carried out by analyzing the DA production of as many isolates as possible. In total, 44 strains of Pseudo-nitzschia spp. were cultured during June and December among which only one strain was found to be toxic. This also supports the argument that toxic P. multiseries does not bloom in a high density in Ofunato Bay. In the case of the causative dino¯agellates of paralytic shell®sh poisoning, nontoxic strains are often observed in toxic species (Yentsch et al., 1978). Similar ®ndings are observed in the DA-producing species. Amphora coaeiformis isolated from Seal River, o Cardigan Bay, Prince Edward Island, is reported to produce DA (Maranda et al., 1990). In contrast, DA was not detected in the strain of the same species from culture collections in the Provasoli-Guillard Center for Marine Phytoplankton (Bates et al., 1989). In the present study, the species identi®cation was carried out only on the strain which produced DA in the culture conditions described above. It is so far not clear whether P. multiseries is included in the rest of the 43 strains, though there is no report on the nontoxic strains of P. multiseries. Species identi®cation of nontoxic strains is under progress.
Acknowledgements We express our sincere thank to Dr. T. Kawamura, Tohoku National Fisheries Research Institute, for his helpful advice in species identi®cation. We also thank Dr. R. Sakai of Kitasato University and Dr. M. Satake of Tohoku University for taking NMR and ESI/MS. This work was partly supported by a grant from the Fisheries Agency of Japan.
References Addison, R.F., Stewart, J.E., 1989. Domoic acid and the eastern Canadian molluscan shell®sh industry. Aquaculture 77, 263±269. Bates, S.S., Bird, C.J., De Freitas, A.S.W., Foxall, R., Gilgan, M., Hanic, L.A., Jhonson, G.R., McCulloch, A.W., Odense, P., Pocklington, R., Quilliam, M.A., Sim, P.G., Smith, J.C., Rao, D.V.S., Todd, E.C.D., Walter, J.A., Wright, J.L.C., 1989. Pennate diatom Nitzschia pungens as the primary source of domoic acid, a toxin in shell®sh from eastern Prince Edward Island, Canada. Can. J. Fish. Aquat. Sci. 46, 1203±1215. Bates, S.S., De Freitas, A.S.W., Milley, J.E., Pocklington, R., Quilliam, M.A., Smith, J.C., Worms, J., 1991. Controls on domoic acid production by the diatom Nitzschia pungens f. multiseries in culture: Nutrients and irradiance. Can. J. Fish. Aquat. Sci. 48, 1136±1144. Douglas, D.J., Bates, S.S., 1992. Production of domoic acid, a neurotoxic amino acid, by an axenic culture of the marine diatom Nitzschia pungens f. multiseries Hasle. Can. J. Fish. Aquat. Sci. 49, 85±90. Garrison, D.L., Conrad, S.M., Eilers, P.P., Waldron, E.M., 1992. Con®rmation of domoic acid production by Pseudonitzschia australis (Bacillariophyceae) cultures. J. Phycol. 28, 604±607. Guillard, R.R.L., Ryther, J.H., 1962. Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt, and Detonula confervacea (Cleve) Gran. Can. J. Microbiol. 8, 229±239.
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