Direct contact between Pseudo-nitzschia multiseries and bacteria is necessary for the diatom to produce a high level of domoic acid
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- Kobayashi, K., Takata, Y. & Kodama, M. Fish Sci (2009) 75: 771. doi:10.1007/s12562-009-0081-5
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Pseudo-nitzschia multiseries loses most of its ability to produce domoic acid when it is cultured axenically. However, domoic acid production recovers when bacteria from the original culture are reintroduced to axenic cultures, indicating a bacterial association with domoic acid production in this species. In order to determine the role of bacteria in domoic acid production, an axenic strain prepared from a nonaxenic strain of P. multiseries was inoculated into media in cellophane tubes, which were then placed in a bottle containing the original nonaxenic culture. Both strains showed characteristic domoic acid production similar to that previously observed. Domoic acid level in the cells of the axenic strain in the tubes was much smaller than that of the cells outside the tubes. These results show that direct contact between living bacteria and P. multiseries is necessary for producing high levels of domoic acid in P. multiseries.
KeywordsCo-culture of axenic and nonaxenic strainsDomoic acidPseudo-nitzschia multiseries
Domoic acid is an excitatory amino acid that is the cause of amnesic shellfish poisoning. This type of shellfish poisoning was first recorded in Prince Edward Island, Canada, in 1987 . The causative phytoplankton of the amnesic syndrome was identified as being several species of diatom belonging to the genus Pseudo-nitzschia [2–5]. Recently, Kotaki et al.  have identified domoic acid in a new species of benthic diatom, Nitzschia navis-varingica, and suggest that domoic acid is distributed widely in diatom species. Many studies have been carried out on the production of domoic acid by Pseudo-nitzschia multiseries, a representative causative species, in batch culture experiments [3, 7, 8]. These studies indicate that environmental bacteria are involved in the production of high levels of domoic acid by P. multiseries, that is, domoic acid production by P. multiseries decreases greatly when the strain is made axenic, while it recovers when certain species of bacteria isolated from the original culture are reintroduced into the axenic culture . Lowered domoic acid production was also observed to recover when bacteria from cultures of other diatom species were reintroduced into axenic cultures . These facts indicate that bacteria involved in domoic acid production by P. multiseries are widely distributed in the natural marine environment. No domoic acid was produced by the bacteria that were involved in the recovery of domoic acid production in P. multiseries, and domoic acid production did not recover after the addition of bacterial extracts to the axenic culture . We report here that direct contact with living bacteria is necessary for P. multiseries to produce a high level of domoic acid.
Materials and methods
Preparation of axenic strain of P. multiseries
A nonaxenic strain of P. multiseries (OFPm033-1) prepared from a single cell collected from Ofunato Bay in 2003 and an axenic strain obtained from the nonaxenic strain described above  by antibiotics treatment were used for the study. Both cultures were kept in antibiotic-free f/2 medium  at 10°C and a light intensity of 50 μmol photons m−2 s−1 with 16:8 h L:D cycle for at least 4 weeks. The medium was changed regularly. In order to assess the bacteria-free condition of the medium, a portion of the axenic culture was inoculated into Marine Broth 2216 (Difco) and STP liquid medium  and incubated for more than 4 weeks to allow bacterial growth. The culture was fixed with formaldehyde and stained with 4′,6-diamidino-2-phenylindole (DAPI; Wako Pure Chemical, Osaka, Japan) according to Porter et al. . The presence of bacteria in the medium was assessed by DAPI staining and fluorescent microscopy.
Growth, chlorophyll a content, and domoic acid production by the axenic strain of P. multiseries
A portion of the antibiotic-treated culture was inoculated into 300 ml of the f/2 medium  in 500-ml Erlenmyer flasks, so that the initial cell density was 3,000 cells/ml, and cultured at 15°C under a light intensity of 100 μmol photons m−2 s−1 with a 16:8 h L:D cycle. During the culture period, 20-ml aliquots were taken from the culture every 3 days for the measurement of domoic acid (14 ml), chlorophyll a (5 ml), and cell growth (1 ml). Domoic acid levels in the cultured cells were analyzed by HPLC using a precolumn fluorescent derivatization method . Growth and domoic acid production of the original unialgal nonaxenic strain were analyzed in the same way and used as the control. All experiments were performed in triplicate. In order to know the survival activity of the cells, 5 ml of cells was harvested from the 20-ml aliquot of the culture during the culture period for the fluorometric analysis of chlorophyll a according to Suzuki and Ishimaru .
Culture of axenic and nonaxenic strains of P. multiseries in the same medium but separated by a cellophane membrane
The axenic strain was inoculated into 200 ml of f/2 medium at a cell density of 3,000 cells/ml. Six 20-ml axenic subcultures were sealed in sterilized cellophane tubes (pore size 5 nm; Viskase Companies, Tokyo, Japan) and suspended in 1.5 l of f/2 medium in a 2-l Erlenmyer flask into which the nonaxenic unialgal culture of P. multiseries had been inoculated to a cell density of 3,000 cells/ml. The two strains, axenic and nonaxenic, separated by a cellophane membrane were cultured at 15°C under a light intensity of 100 μmol photons m−2 s−1 with 16:8 h L:D cycle. A cellophane tube was taken from the culture every 6 days. A small portion of the culture was inoculated into 5 ml of Marine Broth 2216 (Difco), 20% Zobell, and STP medium . A sample of the culture was also inoculated onto agar plates [Marine agar (Difco) and 20% Zobell medium]. These were then incubated at 25°C for 28 days to observe bacterial growth. The remaining cellophane tube culture was used for the analysis of domoic acid and for cell counts under a light microscope in order to record growth. A cellophane tube with 20 ml of sterilized medium was also suspended in the nonaxenic culture as a control and analyzed as for the bacteria-free cultures. Domoic acid and chlorophyll a in the tube-cultured cells were analyzed as described above.
The bacteria-free status of the axenic cultures
When the axenic culture was sampled at exponential, early, and late stationary growth phases, inoculated into Marine Broth 2216 (Difco) and STP liquid medium , and incubated for more than 4 weeks, no bacteria were observed to grow. After DAPI treatment, no staining of nucleic acids was observed either in the medium or on the surface of P. multiseries cells at the various growth stages. This indicated that bacteria were absent in the media and on the surface of the cells.
Growth, chlorophyll a content, and domoic acid production of the axenic strain
Figure 2 shows changes in chlorophyll a levels in the algal cells during culture. Chlorophyll a levels of the axenic strain remained between 2.0 and 3.0 pg/cell during the first 15 days, although the trend towards a slightly increasing level was observed. Over the incubation period, the axenic strain showed the maximum level of domoic acid although the level was much lower than that of the nonaxenic control culture. However, chlorophyll a suddenly disappeared at day 18 coinciding with an absence of domoic acid, suggesting that the cells were dead at day 18. In contrast, chlorophyll a levels in the nonaxenic strain were nearly the same as those of the axenic strain during the first 15 days. After day 15, the level began to decrease slightly, and a level of around 2.0 pg/cell was maintained until day 24 when the experiment was completed. During this period, domoic acid levels in the cell increased. The cells of the axenic strain without chlorophyll a in a stationary phase did not grow when they were transplanted to freshly prepared medium, while those of the nonaxenic strain with pigment grew, supporting the idea that cells of the axenic strain in a stationary phase that have lost pigment are already dead.
Growth, chlorophyll a content, and domoic acid production in the axenic and nonaxenic strains of P. multiseries when cultured in the same medium separated by a cellophane membrane
Chlorophyll a levels in the cells of both algal cultures were comparable to levels when they were cultured separately (Fig. 4). No significant differences in chlorophyll a levels were observed for cultures inside or outside the cellophane membrane during the first 24 days. However, the chlorophyll a level of the axenic strain inside the tube suddenly decreased at day 30, when domoic acid in the cell decreased to an almost nondetectable level, suggesting that most of the cells were dead at day 30.
In contrast, chlorophyll a levels in the nonaxenic strain outside the membrane were maintained up until day 30 (1.3 pg/cell), showing that the cells were surviving during the period, although the level began to decrease slowly at day 24 (Fig. 4). During days 24–30, domoic acid in the cells of the nonaxenic strain increased continuously, although the production rate slowed as chlorophyll a levels decreased.
In a co-culture of P. multiseries in which axenic and nonaxenic strains were separated by cellophane membrane tubes, both strains retained their characteristic domoic acid production profile as observed for individual cultures of the respective strains. That is, no significant domoic acid was detected in cells of the axenic strain, whereas high levels of domoic acid were detected in the nonaxenic strain. These results strongly suggest that direct contact between bacteria and P. multiseries cells is necessary for P. multiseries to produce domoic acid, although the role of bacteria in domoic acid production of P. multiseries is not known.
Axenic cultures of P. multiseries have been reported to produce very low amounts of domoic acid . Thus, it is generally accepted that P. multiseries produces domoic acid autonomously and that bacteria in the medium enhance domoic acid production by P. multiseries. Bates  reported that domoic acid production by axenic strains of P. multiseries is not enhanced by the addition of bacterial extracts with various molecular weights. This finding showed that P. multiseries does not utilize bacterial substances in the medium for domoic acid production.
Pseudo-nitzschia multiseries is also known for being able to remain in the stationary phase for long periods . In the present study, we also observed that cell numbers after maximum growth were maintained for more than 20 days at stationary phase in both axenic and nonaxenic cultures (Figs. 1, 3). However, significant differences in chlorophyll a content were observed for the axenic and nonaxenic strains. In the axenic culture, the level of chlorophyll a began to decrease in the stationary phase and almost disappeared at days 20–30 (Figs. 2, 4), indicating that the cells were dead during the period. In contrast, cells of the nonaxenic strain maintained a constant pigment level for a longer period. These suggest that nonaxenic strains can survive longer than axenic strains under nutrient-deficient conditions. Recently, Bates et al.  reported that several morphotypes of bacteria are attached to the frustule of P. multiseries. They speculate a possible association of these epiphytic bacteria with the enhancement of domoic acid production in the P. multiseries cells, or even autonomous production of domoic acid. Our findings that direct contact with bacteria is necessary for P. multiseries to enhance domoic acid production supports their speculation.
No difference was observed in the domoic acid level between the cell-free media outside and inside the cellophane tubes. Cells of P. multiseries strains are reported to excrete domoic acid into the medium . As shown in Fig. 1, the nonaxenic strain also secreted considerable amounts of domoic acid into the medium, which were comparable to extracellular levels reported by other authors . Thus, domoic acid in the cell-free medium within the tubes is considered to originate from the nonaxenic strain outside the tubes. The lack of domoic acid in cells of the axenic strain clearly showed that P. multiseries did not uptake domoic acid from the medium.
Domoic acid is a small tricarboxylate amino acid that is similar to mugineic acid, a phytosiderophore produced by terrestrial plants . Rue and Bruland  revealed that domoic acid forms chelates with both iron and copper and suggested that domoic acid plays the role of a phytosiderophore in toxigenic Pseudo-nitzschia. However, Maldonado et al.  denied this idea because typical phenomena observed in iron uptake by the organisms involved in siderophores are not observed in domoic acid production by Pseudo-nitzschia. Although we did not analyze iron in the medium or in the cells, the iron concentration in the medium outside and inside the cellophane tubes is considered to be the same as domoic acid in the medium. Nevertheless, domoic acid concentration of the cells in the axenic culture in the cellophane tubes was much lower than that of the nonaxenic culture outside the cellophane tubes. These results indicate that P. multiseries inside the tubes does not endocytose high amounts of domoic acid in the medium. Thus, our results also deny the possible role of domoic acid as a phytosiderophore.
This study is partly supported by a grant-in-aid from the Ministry of Education, and a grant for the Cooperative Research Program of the School of Fisheries Sciences, Kitasato University.