T . Uchida et al. J. Exp. Mar. Biol. Ecol. 241 1999 285 –299
287
Furthermore, the growth of each species in bialgal cultures were simulated using a mathematical model to quantify the relationships between H
. circularisquama and G. mikimotoi.
2. Materials and methods
The strains of Heterocapsa circularisquama HA 92-1 and Gymnodinium mikimotoi GM-1 used were isolated from seawater samples collected from Ago Bay, Japan in
December 1992, and from Hiroshima Bay, Japan in July 1992, respectively. Clonal cultures of these species were obtained by repeated washings using capillary pipettes.
21
The cultures thus obtained were subjected to sterility tests using ST10 medium Ishida
et al., 1986. In both cultures, bacterial growth was not observed. Cultures were maintained at 22618C on a 12-h light dark cycle; illumination was provided by
22 21
cool-white fluorescent lamps at 90–120 mmol photon m s
. Modified SWM3 Itoh and Imai, 1987 was used as the culture medium throughout the experiments. Growth
experiments using bialgal cultures were conducted in 50-ml Erlenmyer flasks with 25 ml of medium. Cells of H
. circularisquama in the logarithmic phase of growth were
21
inoculated at a density of 200 cells ml into cultures of G
. mikimotoi of three different
21
cell densities: 200, 1000 and 2000 cells ml . Growth was measured at 2–5 day
intervals by counting cells in 0.01–0.2-ml culture samples using a Sedgwick–Rafter
21
slide. When cell densities were greater than 20 000 cells ml , the samples were diluted
10–20 times with autoclaved seawater. The condition of the algal cells in the bialgal cultures were observed using an inverted microscope.
Culture filtrates of H . circularisquama and G. mikimotoi were tested in order to verify
the effects of extracellular products on the growth of the other species. Cultures of each
21
species of which the cell density reached 23 000 cells ml for H
. circularisquama, and
21
4600 and 44 000 cells ml for G
. mikimotoi, were passed through membrane filters Millipore RA, pore size 1.2 mm. Then the pH of the filtrate was adjusted to 7.8–8.0
with 1 N HCl. When necessary, it was re-enriched with nutrients at the same concentrations of the modified SWM3. The filtrates thus prepared were sterilized by
passing through a membrane filter Kurabou Steradisc 25, pore size 0.2 mm. Growth experiments were conducted using glassware test tubes with 4.5 ml of the filtrate or
modified SWM3. Culture conditions were the same as stated above. Growth was measured by in vivo chlorophyll a fluorescence using a Turner Designs Model 10-100R
fluorometer Brand et al., 1981.
H . circularisquama and G. mikimotoi were also cultured being separated from each
other with a membrane filter. Half-milliliter cultures of both species were put in each well of 24-well multiplates. The initial cell density of the cultures were 30 000 cells
21 21
21
ml for H
. circularisquama, and 4300 cells ml , 9700 cells ml
, and 29 000 cells
21
ml for G
. mikimotoi. As a control, the flagellate culture was replaced with fresh modified SWM3 medium. Then, a small chamber Millicell PC with a membrane filter
pore size 3 mm fixed to its base containing 0.5 ml of the other species culture was
21
placed into each well. The cell density in this small chamber was 200 cells ml for both
species. Thus we tested if the species outside of the chamber released any inhibitory
288 T
. Uchida et al. J. Exp. Mar. Biol. Ecol. 241 1999 285 –299
substance on the other species cultured in the chamber. These procedures were conducted aseptically throughout the experiments. These cultures were carried out under
the same conditions as the bialgal cultures detailed above. After each set of the cultures, cell morphology was carefully observed each day under an inverted microscope. Then
cell density was determined by counting cells in 0.01–0.1-ml culture samples 4 days after the inoculation.
3. Results
