Diel Vertical Migration and Cell Division of Bloom-Forming Dinoflagellate

Plankton Benthos Res 6(2): 92–100, 2011 Plankton & Benthos Research © The Plankton Society of Japan

Diel vertical migration and cell division of bloom-forming dinoﬂagellate Akashiwo sanguinea in the Ariake Sea, Japan

TOSHIYA KATANO1,*, MAKOTO YOSHIDA2, SOUICHI YAMAGUCHI3, TAKAHARU HAMADA1, KENJI YOSHINO1 & YUICHI HAYAMI1

Ariake Sea Research Project, Saga University, Honjo-machi 1, Saga 840–8502, Japan 1 Present address: Institute of Lowland and Marine Research, Saga University, Honjo-machi 1, Saga 840–8502, Japan 2 Present address: Seikai National Fisheries Research Institute, Fisheries Research Agency, 1551–8, Taira-machi, Nagasaki 851–2213 Japan 3 Present address: Interdisciplinary Graduate School of Engineering Sciences, Kyushu University, Kasuga-Kouen 6–1, Kasuga, Fukuoka 816–8580, Japan Received 2 February 2010; Accepted 7 December 2010

Abstract: Diel vertical migration and cellular division processes of Akashiwo sanguinea were investigated in the Ariake Sea in August 2009. The investigation was carried out in the inner part of the Sea for 24 h from 8:00 on 14 Au- gust. Water samples were collected every 2 hours, and the temperature, salinity, and chlorophyll ﬂuorescence were monitored hourly. To calculate the in situ growth rate, the duration of the cell division was investigated using incubation experiments in a laboratory. In the Ariake Sea, cells of A. sanguinea were abundant in the subsurface layer in the daytime and at a depth of 4 or 5 m at night. In contrast, cells were mostly absent in the deeper layer in the daytime and in the subsurface layer at night. Downward migration seemed to start at 14:00, indicating that dusk is not the cue for migration. A dense patch was formed at a depth of 5 m from 20:00 to 4:00. The highest frequency of dividing cells was recorded at dusk (18:00) in the Ariake Sea and at light-dark transition (circadian time 14) in the laboratory. The estimated duration of cell division was 1.29 h, and the estimated in situ growth rate was 0.45 d1. The present study demonstrated that cells of A. sanguinea actively divided in the deeper layer (3.5 to 5 m) from 18:00. To predict red tide occur- rence by vertically migrating species, the migration pattern and the growth rate in the natural environment should be further clariﬁed for each species. Key words: Akashiwo sanguinea, cell division, diel vertical migration, growth rate

water column stratifies. The vertical migration behavior in Introduction relation to environmental conditions has been studied Akashiwo sanguinea (Hirasaka) Hansen et Moestrup is a mainly in the laboratory (Cullen & Horrigan 1981, Park et representative red tide-causing dinoflagellate in tropical-to- al. 2002). The behavior in the natural environment has also temperate coastal waters (Kiefer & Lasker 1975, Domingos been described. However, the frequency of sampling has to & Menezes 1998, Gómez & Boicenco 2004, Lu & date been insufficient to learn the details of the upward and Hodgkiss 2004). In the Ariake Sea, Western Japan, this mi- downward migration. In addition, the water column depth is croalga dominates during the warm water season and some- different in each system. Thus, detailed information regard- times causes bloom (Yamaguchi unpubl). Recently, Matsub- ing the behavior of A. sanguinea in natural environments ara et al. (2007) reported its growth response to light, tem- should be accumulated for each ecosystem to clarify the perature, and salinity. However, the bloom developmental bloom-development process. process for this alga has not yet been clarified. In order to elucidate the bloom-development process, it This phytoplankter exhibits diel vertical migration be- is essential to have information on the growth of an alga in havior (Kamykowski & Zentara 1977, Cullen & Horrigan the natural environment. However, due to the vertical mi- 1981, Fiedler 1982, Park et al. 2002), which probably plays gration behavior of A. sanguinea, it is quite difficult to eval- a crucial role in the development of the population when a uate its growth in the natural environment using only a bottle incubation experiment. Counting the dividing cells pro- Corresponding author: Toshiya Katano; E-mail, [email protected] vides a substitute for the bottle incubation experiment. The Vertical migration and division of Akashiwo sanguinea 93

Fig. 1. Loss of Akashiwo sanguinea cells by fixation with Lugol solution and Hepes-buffered paraformaldehyde and glutaraldehyde solution (PFA&GA). A non-fixed (live) sample was used as the control. The experiment was carried out in triplicate. Error bars show standard deviations. time series data for the frequency of dividing cells allows Fig. 2. Map showing the sampling station. the calculation of the in situ growth rate without incubation, although this calculation requires the cell division duration M003, Onset, USA). Water samples were collected with a time. Van Dorn water sampler every 2 hours. A portion of the Generally, high numbers of samples should be counted sample was fixed with a fixative (glutaraldehyde 1%, within a day to study diel vertical migration in a natural en- paraformaldehyde 1%, 30 mM Hepes pH 7.4, 0.5 M sucrose vironment. However, due to the difficulty of fixation of this at final concentrations; Katano et al. 2009). At 12:00 and naked dinoflagellate, sample preservation has been almost 18:00 on 14 August and at 0:00 and 6:00 on 15 August, an- impossible. Recently, we developed a new fixative for Chat- other portion was filtered with a dismic filter (pore size tonella cells and found that it was also useful for A. san- 0.45 mm) for the determination of nutrient concentrations. guinea (Katano et al. 2009, Fig. 1). Loss of A. sanguinea Cells of Akashiwo sanguinea in up to 4 mL of the fixed cells by fixation with Hepes-buffered paraformaldehyde sample were counted with a microscope. Frequency of di- and glutaraldehyde solution is negligible, while that with viding cells (Fig. 3) was evaluated with a microscope for Lugol solution is significant. In the present study, we inves- 500 cells, including both dividing and non-dividing cells. tigated the diel vertical migration of a natural population in The nitratenitrite, ammonium, and phosphate concentra- the Ariake Sea using the newly developed fixation protocol. tions were measured with an autoanalyser according to the In the investigation, we evaluated the frequency of dividing manufacturer (SWAAT, BL Tec, Japan). cells to determine the cell division time and to calculate the in situ growth rate. For this calculation, we also conducted Laboratory experiment incubation experiments to estimate the duration of cell divi- A strain (KYAS01) of Akashiwo sanguinea isolated from sion. the Yatsushiro Sea was used for the evaluation of the cell division duration time. An experiment was carried out Materials and methods using SWM3 media (salinity 28) at 26°C under a light condition of 150 mEm2 s1 14 L : 10 D cycle. Temperature and Field observation salinity conditions simulating those of the Ariake Sea dur- The investigation was carried out at station SX2 ing the investigation were used (14–15 August 2009). Du- (N33°0601, E130°1224, Fig. 2) starting at 8:00 on 14 plicate cultures in the exponential phase were used for the August 2009. Water depth at the station ranges between 4 experiment. A portion of the sample was collected at 2- and 9 m at spring tide. The water temperature, salinity, and hour intervals from circadian time 2 (CT2) for 24 h. Sam- chlorophyll fluorescence were measured every hour using ples were immediately fixed as reported above. At least 500 AAQ1183 (JFE Advantech, Japan). The Secchi depth was cells were evaluated for the determination of the frequency also measured in the daytime. Solar irradiance was moni- of dividing cells for each sample. tored at 10-second intervals from 6:10 on 14 August to 8:50 on 15 August using a quantum sensor from a boat (S-LIA- 94 T. KATANO et al.

Fig. 3. Micrographs of Akashiwo sanguinea cells. Panels a, b, and c, live cells of strain KYAS01 (a, b, and c); ﬁxed cells of strain KYAS01 (d, e, and f); and ﬁxed cells of the natural population collected at a depth of 3.5 m at 18:00, 14 August 2009 (g, h, and i). Bars in panels indicate 20 mm. Cells in panels a, b, d, e, g, and h were counted as dividing cells and cells in panels c, f, and i were counted as non- dividing cells.

Ariake Sea and the td, the in situ growth rate was calcu- Calculation lated. Samples for the evaluation of frequency of dividing The growth rate (m ) was calculated from the fraction of cells were determined by the vertical distribution of chloro- cells undergoing mitosis as follows (McDuff & Chisholm phyll fluorescence in most cases (Table 1). 1982): l n μ ln(lfi ) , Results ntd ∑ il Physical and chemical environments during the field ob- where fi is the fraction of cells undergoing mitosis, n is the servation sample number for a 24-h period, and td is the duration of mitosis. We applied this equation assuming dividing cells to Weather during the investigation was fair in the daytime be cells undergoing mitosis. The growth rate was calculated and rainy at night. Solar radiation reached 2000 mEm2 s1 from changes in cell density during the incubation experi- at noon (Fig. 4). Sunset on 14 August was at 19:06, and ment as follows: sunrise on 15 August was at 5:41. The water temperature l ranged from 26.4 to 29.9°C, and the water column was μ ln(Nf / N0 ) , thermally stratified (Fig. 5). From 2:00 to 4:00 on 15 Au- t gust, the salinity at the surface suddenly decreased from where N0 and Nf are the cell densities at time zero and after 28.7 to 25.4 (Fig. 5), probably due to the freshwater input the incubation, respectively, and t is the incubation period from rivers as a result of heavy rain during the investigation of one day. From the incubation experiment, we calculated period. Indeed, the Acoustic Doppler Current Profiler td. Moreover, from the frequency of dividing cells in the (ADCP) at the station revealed that a front passed at the Vertical migration and division of Akashiwo sanguinea 95

Table 1. Depth of the samples for the determination of frequency of dividing cells shown in Fig. 9.

Date Time Depth

14 Aug. 2009 8:00 2 m 10:00 1.5 m 12:00 1.4 m 14:00 1.5 m 16:00 3 m 20:00 4.5 m 22:00 4 m 15 Aug. 2009 0:00 4 m 2:00 5 m 4:00 5 m 6:00 3 m 8:00 1.5 m

Fig. 4. Changes in solar radiation during the investigation period at the sampling station.

same time (data not shown). The nitrate nitrite concentration did not show any trends in the vertical profile (Fig. 6). At 12:00 on 14 August, the nitrate nitrite concentration was the highest at the surface, while the vertical profile was bimodal at 18:00 on 14 August. The concentration of Fig. 5. Diel changes in the vertical profile of temperature (a), 1 nitrate nitrite was relatively low (0.43–2.21 mmol N L ) salinity (b), chlorophyll fluorescence (c), and abundance of 1 compared with that of ammonium (0.37–6.30 mmol N L ). Akashiwo sanguinea (d). The phosphate concentration was high (1.1 mmol P L1) and was apparently not a limiting factor for growth of the and bacillariophyceans, Chaetoceros and Thalassiosira. alga. Cells of A. sanguinea were abundant at depths between 1 and 3 m in the daytime (Figs 5, 7). The cells were uniformly Diel vertical migration of Akashiwo sanguinea in the distributed in the whole water column at 18:00. From Ariake Sea 20:00, high numbers of cells were found at 5 m, where the The cell density of Akashiwo sanguinea ranged from 0 to highest value was recorded at 2:00. This vertical distribu- 130 cells mL1 (Fig. 5). Dominant taxa other than A. san- tion continued until 4:00. At 6:00, the cell density increased guinea observed in the samples were the raphidophycean, at 3 m (Figs 5, 7), indicating that the upward migration had Chattonella; dinophyceans, Prorocentrum and Ceratium; already started. The next morning (8:00, 15 August), most 96 T. KATANO et al.

Fig. 6. Changes in the vertical distribution of NO2 NO3-N (white circles), NO2 NO3 NH4-N (black circles), and PO4-P (white squares) concentrations. NH4-N concentration was calculated as the difference between black- and white circles.

Fig. 7. Changes in the vertical distribution of temperature (solid lines) and cell abundance of Akashiwo sanguinea (solid lines with black circles). The Secchi depth is indicated in each panel. Vertical migration and division of Akashiwo sanguinea 97

Fig. 9. Changes in frequency of dividing cells of Akashiwo sanguinea in the Ariake Sea.

It should be noted that the frequency of dividing cells could not be evaluated at the peak depth in the vertical distribution in some cases (Table 1, Fig. 7). As mentioned above, samples for the evaluation of the frequency of divid- Fig. 8. Changes in the cell density of Akashiwo sanguinea ing cells were determined by the vertical distribution of (upper panel) and changes in the frequency of dividing cells chlorophyll fluorescence in most cases (Table 1, Fig. 5). At (lower panel). Data are from the laboratory experiment. Error bars 16:00 and 18:00 on 14 August and at 6:00 on 15 August, indicate the standard deviation of triplicate replications. Bars on we collected water samples at 3 m (16:00, 14 August), 3.5 the upper panel indicate light and dark periods. m (18:00, 14 August), and 3 m (6:00, 15 August) since chlorophyll fluorescence did not show distinct peaks in the vertical distributions (Fig. 5). During the nighttime, chloro- cells (95.2% of cells in the water column) were distributed phyll fluorescence showed a bimodal distribution; high val- at depths between 0 and 1.5 m. ues were detected at depths of 0–2 m and 4–5 m (Fig. 5). In these cases, we collected water at the deeper peaks for the Frequency of dividing cells of Akashiwo sanguinea in the estimation, since the cell density was high. At 14:00 on 14 laboratory experiment August, maximum chlorophyll fluorescence was detected at 1.5 m; however, the cell density was highest at 3 m (Figs 5, The frequency of dividing cells in the laboratory experi- 7). As a result, at 14:00 and 18:00 on 14 August and at 6:00 ment was high at CT12 and CT14 (4.04 and 4.99%, respec- on 15 August, we could not evaluate the frequency of divid- tively; Fig. 8). The value decreased after CT14. Desmos- ing cells at the peak depth of the vertical distribution. chitic (Pfiester & Anderson 1987) cell division was observed during the dark period. Dividing cells, observed in the samples obtained from laboratory and the Ariake Sea, Discussion are shown in Fig. 3 (Fig. 3a, b, d, e, g, and h). The oblique Accurate measurement of the in situ growth rate of algal division started with splitting at the sulcus. By late division, species exhibiting vertical migration is essential for the pre- cells were loosely attached at the epicone of the daughter diction of population development. The vertical migration cell. The growth rate was 0.35 d 1 from the changes in the behavior is also an important process that appears to play a cell density during a 24-h cycle. The duration of cell divi- crucial role in the growth, movement, and possibly initia- sion was calculated as 1.29 h. tion of blooms. However, neither the in situ growth rate nor detailed information on vertical migration, such as the tim- Frequency of dividing cells of Akashiwo sanguinea in the ing of the ascending and descending migrations and the sea and the in situ growth rate vertical positioning in the natural environment, have yet The frequency of dividing cells in the Ariake Sea peaked been described for Akashiwo sanguinea. The present report at 18:00 and gradually decreased until 22:00 (Fig. 9). In the is the first in which the in situ growth rate is estimated and daytime, dividing cells were not detected in most cases. Our the diel vertical migration pattern in a natural environment results clearly demonstrate that the cell division of has been studied. Although the data shown in the present Akashiwo sanguinea was well synchronized. Cell division study are from one field survey, our sampling was done in also occurred from 18:00 to 22:00 in the deeper layer. the bloom-development period. Indeed, the A. sanguinea Using the duration of 1.29 obtained from the laboratory ex- population formed a red tide in late September in the Sea periment, the in situ growth rate was calculated as 0.45 d 1. (data not shown). Thus, our data may provide important in- 98 T. KATANO et al. sight into bloom development. tion cell cycle is synchronized. This estimation is quite sim- Accurate estimation of the in situ growth rate using the ple and possibly accurate, although 2-hour interval sam- frequency of dividing cells depends on three assumptions: pling is quite laborious and sometimes dangerous in natural (1) the duration of cell division is constant under varying environments. Further information should be accumulated conditions; (2) the duration is identical for all cells in a to generalize the cell division pattern and the relationship population; and (3) all cells in a population are active between the frequency of dividing cells at dusk and the (Campbell & Carpenter 1986, Tsujimura 2003). Tsujimura daily growth rate. (2003) found that the frequency of dividing cells of a Downward migration started 5 hours before sunset (Figs cyanobacterium, Microcystis, did not become 0 during the 5, 7), suggesting that dusk is not the cue for the descending 24 h of the experiment. He suggested that the minimum migration in A. sanguinea. At 14:00, the peak in cell abun- fraction of dividing cells during a 24-hour cycle corre- dance in the vertical profile (3.5 m) was twice as deep as the sponds to the portion of inactive cells (Tsujimura 2003). Secchi depth (1.8 m; Fig. 7). Cullen & Horrigan (1981) However, this was not the case for the present study. The thoroughly studied the diel vertical migration behavior of A. fraction of dividing cells became 0% during the daytime, sanguinea in a laboratory tank. In their data, the peak in cell suggesting that the dividing cells observed at dusk had di- abundance did not change until at least 15:00, 4 hours be- vided before dawn. fore sunset, although downward migration was detected 1 The duration of nuclear division of Ceratium is affected hour before sunset. Kamykowski et al. (1998) demonstrated by temperature (Weiler & Eppley 1979). The duration in- that cells of Karenia brevis (Davis) Hansen et Moestrup ( creases under lower temperature conditions (Weiler & Epp- Gymnodinium breve Davis) in the surface layer showed ley 1979), causing an error in the estimation of the growth lower protein and carbohydrate contents than did cells in the rate. In the present study, the experiment was conducted deeper layer, suggesting the presence of a biochemical influ- under the same temperature conditions as those in the envi- ence on the vertical migration behavior. Such an intercellu- ronment in order to prevent such an error. Therefore, the ef- lar condition can be a cue for the downward migration for A. fect of temperature on the duration is inconsequential in the sanguinea, although precise information is still unavailable. estimation of the growth rate in the present study. However, In the daytime, cells of A. sanguinea did not form a Weiler & Eppley (1979) also pointed out that the duration patch at the surface (Figs 5, 7). Surprisingly, cells were not of nuclear division was shorter than that in the natural envi- found at the surface at 12:00 (Fig. 7). Similar vertical posi- ronment. Currently, we have insufficient information to tioning in the daytime has been reported for this species reach a conclusion regarding nuclear division; however, the (Cullen & Horrigan 1981, Fiedler 1982). Cullen & Horri- duration of cell division under various conditions, such as gan (1981) reported that A. sanguinea migrated to the sub- different nutrient concentrations and/or light intensities, surface layer when nitrate was depleted under an irradiance should be further evaluated. of 250 mEm2 s1, which is close to the saturating intensity Although the estimation of growth rate from the fre- required for its photosynthesis. Ault (2000) demonstrated quency of dividing cells includes several issues likely to that the dinoflagellate Prorocentrum triestinum Schiller mi- cause error, an advantage is that this method does not re- grates vertically to maximize its photosynthetic rate. The quire incubation. The vertically migrating species move be- maximum photosynthetic rate of the alga occurred at a tween two layers within a day, when light and nutrients are depth of 0.5 m, where the cell density was also the highest vertically separated (Cullen & Horrigan 1981). In such a (Ault 2000). These cells may respond to the environmental situation, it is difficult to measure growth rate using bottle light conditions. incubation experiments. Thus, we believe that this tech- Another important finding of the present study is that the nique is useful for the estimation of the growth rate of ver- cells remained at a depth of 5 m in the nighttime (Figs 5, 7). tically migrating dinoflagellates or raphidophytes. It has already been revealed that A. sanguinea can swim The results of the present study demonstrate that A. san- through large temperature gradients (Kamykowski & Zen- guinea cells divided at a depth of 3.5–5 m during the night- tara 1977, Cullen & Horrigan 1981) and the cells accumu- time (Fig. 5). The in situ growth rate was calculated as 0.45 late below the thermocline in the nighttime, which is con- d1, suggesting that the A. sanguinea population increased sistent with our results (Figs 5, 7). However, these cells did 1.56 times in cell density during the investigation. The not migrate to the bottom of the water column. A similar value was higher than that observed in the laboratory (0.35 vertical migration pattern had been already observed in Au- d1). Although the reason for this result is currently un- gust 2005 in Isahaya Bay of the Ariake Sea (Yamaguchi un- known, the natural population seemed to develop actively publ). Hourly CTD observations revealed that a high- during the investigation. chlorophyll patch appeared at depths between 5 and 7 m The frequency of dividing cells reached a maximum at during the nighttime and between 1 and 2 m during the day- dusk (18:00) in the Ariake Sea (Fig. 9) and at the light-dark time when the A. sanguinea bloom occurred. transition (CT14) in the laboratory (Fig. 8), which suggests The migrating depth during the nighttime appears to be that the frequency of dividing cells at dusk can be an indi- important for the uptake of nutrients at depths where sur- cator of the in situ growth rate, assuming that the popula- face nutrients are exhausted. For example, Chattonella spp., Vertical migration and division of Akashiwo sanguinea 99 which also cause blooms in the Ariake Sea, can migrate to tific Research (C) (20589002) from the Japan Society for a depth of 7.5 m during the nighttime to take up nutrients the Promotion of Science. We appreciate Prof. G Ko- (Watanabe et al. 1995), while A. sanguinea could not mi- bayashi’s support in the experiments. grate to a depth greater than 5 m (Figs 5, 7). When nutrients are available at depths greater than 6 m, Chattonella can ef- References ficiently utilize such deep nutrient sources to develop its population. On the other hand, in such a case, A. sanguinea Ault TR (2000) Vertical migration by the marine dinoflagellate hardly uses such deep nutrient sources. Therefore, the verti- Prorocentrum triestinum maximises photosynthetic yield. Oe- cal profile in the nutrient concentration and the migrating cologia 125: 466–475. depth during the nighttime are important to predict the Bearon RN, Grunbaum D, Cattolico RA (2006) Effects of salinity blooming species in the environment. structure on swimming behavior and harmful algal bloom for- The nutrient concentration showed no obvious trend in mation in Heterosigma akashiwo, a toxic raphidophyte. Mar the vertical profile, however (Fig. 6). At 0:00 on 15 August, Ecol Prog Ser 306: 153–163. the ammonium concentration tended to be high in the Campbell L, Carpenter EJ (1986) Diel patterns of cell division in deeper layer, but the concentration at 4.5 m was 2.01 mmol marine Synechococcus spp. (Cyanobacteria): use of the fre- NL1. In addition, the nitrate + nitrite concentration was quency of dividing cells technique to measure growth rate. Mar Ecol Prog Ser 32: 139–148. high in the surface layer at 12:00 on 14 August and at 6:00 Cullen JJ, Horrigan SG (1981) Effects of nitrate on the diurnal on 15 August. Since we did not examine the nutrient up- vertical migration, carbon to nitrogen ratio, and the photosyn- take, it is uncertain whether A. sanguinea cells received any thetic capacity of the dinoflagellate Gymnodinium splendens. benefit from the nocturnal uptake of inorganic nitrogen. 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