J. Phycol. 36, 1057–1060 (2000)
DOMOIC ACID PRODUCTION IN NITZSCHIA SP. (BACILLARIOPHYCEAE) ISOLATED FROM A SHRIMP-CULTURE POND IN DO SON, VIETNAM 1 Yuichi Kotaki, Kazuhiko Koike School of Fisheries Sciences, Kitasato University, Sanriku, Iwate 022-0101, Japan
Makoto Yoshida Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi, Bunkyo, Tokyo 113-8657, Japan
Chu Van Thuoc, Nguyen Thi Minh Huyen, Nguyen Chu Hoi Haiphong Institute of Oceanology, Da Nang Street, Haiphong, Vietnam
Yasuwo Fukuyo Asian Natural Environmental Science Center, The University of Tokyo, Yayoi, Bunkyo, Tokyo 113-8657, Japan
and Masaaki Kodama2 School of Fisheries Sciences, Kitasato University, Sanriku, Iwate 022-0101, Japan
et al. 1990, Garrison et al. 1992, Lundholm et al. 1994, Míguez et al. 1996, Rhodes et al. 1996, Vrieling et al. 1996, Trainer et al. 1998, Kotaki et al. 1999). In addition to Pseudo-nitzschia spp., DA has been reported in the benthic diatom Amphora coffeaeformis (Agardh) Kützing (Shimizu et al. 1989, Maranda et al. 1990). During a survey of algal species involved in the mass mortality of shrimp larvae in Asian countries, we found pennate diatom species in phytoplankton samples collected in a shrimp-culture pond at Do Son, near Haiphong, Vietnam. We report here DA production by the diatom, which is described as a new species belonging to Nitzschia in a companion paper (Lundholm and Moestrup 2000).
Domoic acid (DA), a neuroexcitatory amino acid, was detected in batch culture of the newly recognized species Nitzschia navis-varingica Lundholm et Moestrup. The production of DA by this diatom was confirmed by electrospray ionization mass spectrometry. The diatom was collected from a shrimp-culture pond in Do Son, Vietnam. The production of DA (1.7 pg·cell⫺1) is within the levels reported for Pseudo-nitzschia multiseries (Hasle) Hasle. The DA production started during the late exponential growth phase and reached a maximum during the early stationary growth phase. Maximum DA levels in the axenic culture decreased to about half that of the nonaxenic culture (0.9 pg·cell⫺1 vs. 1.7 pg·cell⫺1), suggesting that DA production by the new species is influenced by bacteria.
materials and methods Isolation and culturing. Phytoplankton samples were collected by net hauling (mesh size of 20 m) from a shrimp-culture pond at Do Son (20⬚43⬘N, 106⬚47⬘E) in September 1997. The pond is about 20 ⫻ 50 m square, 50 cm deep, and located about 150 m inland from the coast. The pond is lined by mangrove trees and is connected to the coast by a channel. Diatom cells were isolated by the capillary method and inoculated singly into f/2 medium (Guillard 1983). The isolates obtained were cultured in 30 mL f/2 in culture tubes maintained at 20 ⬚ C under a photon flux density of 120 mol photons·m⫺2·s⫺1 in a 16:8 light:dark regime. An isolate (VSP974-1) showing a peak corresponding to DA in the HPLC was cultured to an amount of 3 L under the same conditions for further chemical analysis of DA. Analysis and identification of DA. Two weeks after the beginning of the stationary growth phase, when DA production of P. multiseries has been reported to increase (Bates et al. 1991), the amount of DA in the culture was analyzed by HPLC with precolumn derivatization according to Pocklington et al. (1990). The DA-like substance was purified by successive chromatographies on Wakosil 25C18 (Wako Pure Chemicals, Ltd., Osaka, Japan, 10 ⫻ 470 mm) and Develosil ODS-5 (Nomura Chemical Co., Ltd., Seto, Japan, 8.0 ⫻ 250 mm). The purified substance was dissolved in 50% methanol and analyzed by electrospray ionization mass spectrometry (ESI MS) using a spectrometer (model JMS-700, JEOL, Tokyo, Japan).
Key index words: amnesic shellfish poisoning; benthic diatom; domoic acid; Nitzschia sp.; Vietnam Abbreviations: DA, domoic acid; ESI MS, electrospray ionization mass spectrometry Amnesic shellfish poisoning caused by domoic acid (DA), a neuroexcitatory amino acid, was first observed on Prince Edward Island, Canada in 1987 (Wright et al. 1989). The causative organism in the Canadian case was identified as a pennate diatom Pseudo-nitzschia multiseries (Hasle) Hasle (formerly Nitzschia pungens f. multiseries), which had bloomed in the area (Bates et al. 1989). Since the first incidence in Canada, DA production has been reported in P. multiseries from other parts of the world and in other Pseudo-nitzschia species (Martin
1Received 2Author
22 November 1999. Accepted 3 August 2000. for correspondence: e-mail
[email protected].
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DA production in axenic and nonaxenic culture. Cells of the DAproducing strain (VSP974-1) were incubated for 2 days in f/2 containing a mixture of antibiotics (final concentration in the culture: gentamycin 0.05 mg·mL⫺1, penicillin-G 1.6 mg·mL⫺1, streptomycin 0.8 mg·mL⫺1) (Wako Pure Chemicals, Ltd.) under the conditions mentioned above. Cells were then isolated by a capillary, washed 3 times with sterilized medium, and inoculated into new medium to establish an axenic culture. The axenic condition was examined by checking for bacterial growth after incubation at 25⬚ C for 4 weeks using both agar plates and liquid media of STP (Provasoli et al. 1957) and Marine Broth 2216 (Difco, Detroit, MI). The absence of bacteria was also confirmed by DAPI staining of the culture (Porter and Feig 1980). To compare the DA productivity, both axenic and the nonaxenic strains of VSP974-1 were cultured in f/2 medium at 25 ⬚ C under a photon flux density of 120 mol photons·m⫺2·s⫺1 in a 16:8 light:dark regime. One-liter cultures in 2-L flasks were inoculated with cells from a 6-day-old culture to an initial cell density of about 2000 cells·mL⫺1. Cell concentration was determined by microscopic cell counts using a Sedgewick-Rafter counting chamber (Rigosha Co., Ltd., Tokyo, Japan) each day for the first 10 days and every 2 days thereafter. Growth rate was calculated from the increase in cell number during the exponential growth phase according to Fukazawa et al. (1980). A 10-mL aliquot of the culture was collected every 2 days and extracted for HPLC analysis of DA in the whole culture (cell plus medium). A second 10-mL aliquot was filtered through a mixed cellulose ester membrane filter with a pore size of 0.5 m for DA analysis of the cell-free media. DA concentration per cell was calculated from the total cell number and the difference of DA concentration between the whole culture and the cell-free medium.
results Cultures. Two different species of pennate diatoms were observed in the samples. One of the species was identified as a new species belonging to the genus Nitzschia, as described in the accompanying paper
(Lundholm and Moestrup 2000). The other species was unidentified. Five monoclonal cultures were established for each species. Identification of DA. All isolates of the new Nitzschia species showed a peak at the retention time identical to DA in the HPLC analysis, whereas those of the second species did not. One of the positive isolates (VSP974-1) was therefore cultured to 3 L. The amount of the putative DA in the cells and the culture medium at day 15 was estimated by HPLC to be 64.3 ng·mL⫺1 and 10.3 ng·mL⫺1, respectively. The compound subsequently purified from the cell extract shows an ion peak at m/z 312 in ESI MS that is assignable to [M ⫹ H]⫹ of DA (cf. Kotaki et al. 1999), indicating that the Nitzschia species produces DA (Fig. 1). DA production in axenic and nonaxenic culture. Bacteria were not observed when the antibiotic-treated culture was inoculated onto agar plates or into liquid media of STP (Provasoli et al. 1957) and Marine Broth 2216 (Difco) and incubated for 4 weeks. Bacteria were also not observed when the culture was stained with DAPI and examined microscopically. Both methods indicate that the antibiotic-treated culture was axenic. When the axenic and the nonaxenic culture were maintained under identical growth conditions, the nonaxenic culture reached the stationary phase at day 4, whereas the axenic culture reached the stationary phase at day 7 (Fig. 2A). The former showed a higher growth rate (1.53 divisions·d⫺1) than the latter (0.92 divisions·d⫺1). The maximum cell yield of both cultures was approximately the same. For the first 2 to 4 days of the experiment, the levels of DA per cell decreased in both cultures (Fig. 2B). This experiment (Fig. 2) was repeated and similar results (not shown) were obtained.
Fig. 1. ESI MS of the DAlike substance purified from Nitzschia sp. (VSP974–1).
DOMOIC ACID IN A NEW NITZSCHIA SPECIES
The level of DA per cell began to increase during the late exponential phase in both the axenic and the nonaxenic culture at days 4 and 6, respectively. The maximum amount of DA per cell in the axenic culture was approximately half that of the nonaxenic culture (0.9 pg·cell⫺1 vs. 1.7 pg·cell⫺1). The level of DA per cell of both cultures decreased during the late stationary phase (Fig. 2B). Low levels of DA were detected in the cell-free medium of both nonaxenic and axenic cultures, increasing slowly from the beginning of the experiment into the exponential and stationary growth phases. discussion The results confirm that a new pennate diatom species (Lundholm and Moestrup 2000) produces DA. Maximum cellular DA in the nonaxenic culture (1.7 pg·cell⫺1) was within the levels reported for P. multiseries (0.1–21.0 pg·cell⫺1) and P. seriata (Cleve) Peragallo (0.3–33.6 pg·cell⫺1), considerably more than that of P. turgidula (0.03 pg·cell⫺1) but less than that of P. australis Frenguelli (12.0–37.0 pg·cell⫺1) (see Bates 1998). DA production of our strain began during the late exponential phase. The cellular DA level reached a maximum at a relatively early stationary phase and then declined. The amount of DA per cell observed early in our experiment is likely due to the presence of DA within the cells (6 days old) used to inoculate the culture, although we did not measure their DA
Fig. 2. Cell growth (A) and DA production (B) of axenic and nonaxenic cultures of Nitzschia sp. (VSP974–1).
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amounts. As the cell number increased during the early exponential phase, the DA per cell decreased until the cells began to produce DA de novo (see Douglas and Bates 1992). The amount of DA in the medium increased slowly from the beginning of the experiment, indicating that DA leaked from the cells to the surrounding medium continuously. DA production by Pseudo-nitzschia species, especially P. multiseries, has been studied intensively in laboratory experiments. These studies indicate that DA is mainly produced during the stationary growth phase (Subba Rao et al. 1990, Bates et al. 1991, 1993, 1995, Douglas and Bates 1992, Wohlgeschaffen et al. 1992, Douglas et al. 1993, Lewis et al. 1993). However, small amounts of DA can be detected during the late exponential phase both in the Nitzschia studied here and in some species of Pseudo-nitzschia (Garrison et al. 1992, Lundholm et al. 1994, Pan et al. 1996). The changes in DA production during the growth phase in P. multiseries batch culture led Bates (1998) to hypothesize the existence of “at least two different, but perhaps related, triggering mechanisms for DA production.” Interestingly, the high levels of DA produced during the late stationary phase of P. multiseries are observed only in nonaxenic cultures; axenic cultures produce substantially less DA. Douglas et al. (1993), followed by Bates et al. (1995), reported that DA production of axenic culture of P. multiseries decreased to 1/20 or less than that of nonaxenic cultures. The reintroduction of bacteria to the axenic culture of P. multiseries enhanced the DA production by 2- to 115-fold, indicating that at least one of the triggering mechanisms of DA production involves the environmental bacteria. The level of DA in the axenic culture of our species decreased to about half that of the nonaxenic culture, suggesting that DA production of our new Nitzschia is also influenced by environmental bacteria. However, the bacterial influence on the DA production of our species was not as obvious as observed in P. multiseries (Douglas et al. 1993, Bates et al. 1995). Although our strain was obtained from a phytoplankton sample collected from the water column, the cells weakly adhered to the bottom of the culture flasks and subsequently clumped together during the culture period, suggesting that the species may be benthic. There is little information about DA production in diatom species other than those belonging to the genus Pseudo-nitzschia. The benthic diatom A. coffeaeformis, which was isolated from toxic mussels in Canada, is also reported to produce low levels of DA (Shimizu et al. 1989, Maranda et al. 1990). This observation, combined with the present results, indicates that DA is present in additional diatom species around the world. The results also show that we have to be aware of the potential for amnesic shellfish poisoning during blooms of diatoms other than just Pseudo-nitzschia. Note added in proof: Purity of the purified DA-like substance was confirmed by a high performance silica gel TLC with a solvent system (1-butanol : acetic acid : water ⫽ 7 : 2 : 3) visualized
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by ninhydrin and sulfuric acid. The substance showed a single peak at the retention time of DA in HPLC with a Develosil ODS-5 column (mobile phase: 10% acetonitrile in 20 mM phosphate buffer, pH 2.5; detection: absorbance at 242 nm). UV absorption of the purified substance gave a typical spectrum of DA, showing a peak at 242 nm. We thank Dr. R. Sakai, associate professor of Kitasato University, for taking an ESI mass spectrum. Supported in part by a grant-in-aid for international scientific research (field research) from the Ministry of Education, Science, Sports and Culture of Japan.
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