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GSTF International Journal of BioSciences (JBio) Vol.2 No.1, December 2012 Diel cell cycle analysis of Heterosigma akashiwo (Raphidophyceae) with special reference to vertical migration behavior Juyun Lee1, Myung-Soo Han2* and Man Chang1* 1 Marine Ecosystem Research Division, Korea Institute of Ocean Science and Technology (KIOST), 787, Haeanro, Ansan 426-744, Korea 2 Department of Life Science, Hanyang University, 133-791 Seoul, Korea * Corresponding author E-mail: [email protected] Tel: +82-31-400-6468; Fax: +82-31-400-6495 been widely applied in the eco-physiological study of algal plankton (Li 2002; Marie et al. 1997; Olson et al. 1985; Olson et al. 2003). One useful application of flow cytometry is for diel cell cycle analysis (Marie et al. 1997) including changes that occur during a full light-dark cycle. Cells of Amphidinium operculatum divide at night before the transition from dark to light (Leighfield and Van Dolah 2001), while Karenia brevis cells start to divide 2 h before the dark-light transition, and continue 6 h after the transition (Van Dolah et al. 2008). Kohata and Watanabe (1986) investigated the changes in H. akashiwo cell size, which indirectly equates to cell division. Based on that information, they hypothesized that the cell division occurred near the dark-light transition in the laboratory. They also found that the decrease in cell size and upward migration occurred simultaneously in the morning, suggesting synchronous division with the vertical migration. Generally, the cell cycle is composed of 4 phases with respect to the DNA content of each cell. This phase is preceded by G1-phase which is the first gap stage before the DNA synthesis which named Sphase. After S-phase, The G2-phase is the second gap, between the S-phase and mitosis which named Mphase of cell cycle (for review see Chisholm 1981). Regardless of whether diel cell cycle is synchronous or not, Smith and Dendy (1962) showed that the specific growth rate can be calculated from the fraction and the duration of the M-phased population. And McDuff and Chisholm (1982) established an equation for estimating the daily mean growth rate in phytoplankton. Diel cell cycle analysis has also been applied to measure the growth rate of oceanic picophytoplankton (Campbell and Carpenter 1986). Since this analysis does not require incubation, it is appropriate for the measurement of microalgae growth when vertical 1. INTRODUCTION The marine alga, Heterosigma akashiwo(Raphidophyceae), is known to frequently form blooms in temperate coastal waters throughout the world (Horner et al. 1997; Smayda 1998; Taylor and Haigh 1993; Taylor and Horner 1994). H. akashiwo populations usually develop rapidly in early summer (Imai 2000; Shikata et al. 2007), forming dense blooms in the upper water column at or near the surface (Handy et al. 2005; Honjo 1993; Yamochi and Abe 1984). Its blooms can cause massive fish kills through the production of chemicals such as neurotoxins, cardiotoxins, and reactive oxygen (Endo et al. 1992; Khan et al. 1997). In addition to the significant impacts to fish populations, algal blooms can cause a decrease in plankton biodiversity and are sometimes accompanied by low oxygen in deeper water (Tsutsumi 2006) which may kill shellfish and other invertebrates. Given these potential impacts, a better understanding of the conditions leading to an algal bloom, and the bloom process itself, are important in helping to protect coastal ecosystems. The accumulation of algae on the surface water is caused by biological processes such as growth and vertical migration (Bearon et al. 2006) and/or physical processes such as wind-driven advection (Hill 1991). These events should be considered separately in the analysis of the bloom-development process. Since cells of H. akashiwo can accumulate in “nutrientdepleted” surface waters during the daytime and move downward to “nutrient-rich” bottom waters at night (Watanabe et al. 1983; Watanabe et al. 1988), growth rate measurement for this species is quite difficult when employing in situ incubation experiments using bottles. To study the biological processes of a H. akashiwo bloom, we used flow cytometry which has DOI: 10.5176/2251-3140_2.1.19 30 © 2012 GSTF GSTF International Journal of BioSciences (JBio) Vol.2 No.1, December 2012 migration is a factor. Recently, this technique was also applied to the dinoflagellate Karenia brevis (formerly Gymnodinium breve), which is the cause of toxic red tides (Van Dolah and Leighfield 1999; Van Dolah et al. 2008). Van Dolah et al. (2008) investigated the growth rate of K. brevis in natural environments over five years and found that the growth rate varied from 0.17 to 0.55 day-1; however, the timing of cell division was apparently not dependent on the growth rate. For H. akshiwo, this cell cycle analysis has not yet been applied to natural populations. The cell membrane of H. akashiwo is generally not well preserved by fixation probably due to osmotic changes. Moreover, the cells sometimes form aggregates after fixation. Therefore, it is hard to apply flow cytometry for H. akashiwo. Recently, we found that a fixative using sodium cacodylate works well with H. akashiwo cells, and have used it (Lee et al. 2012), in the present study to investigate the diel cell cycle with flow cytometry. This study was thus carried out to clarify the cell cycle and pattern of vertical migration to enhance the bloom formation and furthermore the relation between diel vertical migration and the cell cycle. analysis was performed on a FACS Calibur (BD Biosciences, San Jose, CA) equipped with an aircooled argon laser (488 nm, 150 mW). Histograms of the relative DNA amount were analyzed using the Modfit program (Verity Software House, Inc., Palo Alto, CA, USA) to quantify the percentage of cells in each of the growth stages (G1, S, G2+M). At least 10,000 cells were analyzed. Using the sum of the S- and G2+M-phase fractions, the daily mean specific growth rate, µS+G2M, was calculated according to the following equation from Chang and Carpenter (1991); Where (tS)i is the sampling interval of sample i, measured in hours. We used the combined S, G2, and M-phases as the terminal event, and measured the duration term (Tx) by monitoring the diel change of the S- and G2+M-phase fractions [fs(t) and fG2M(t)]. 2.4. Counting H. akashiwo cells were fixed with Lugol’s solution for counting. An aliquot of the preserved sample was placed in a Sedgewick-Rafter counting chamber and cells were enumerated under a light microscope (Axioplan, ZEISS, Germany). 2. METHODS 2.1. Cultures A cyst of H. akashiwo was isolated with a capillary pipette on June 2005 from Masan Bay, located in the southern part of Korea. The cyst was incubated in F/2 media (Guillard and Ryther 1962) at 20oC under a 12:12 h light:dark cycle. A unialgal culture established from the germinated cyst was tentatively named as strain HYM06HA (Ki and Han 2007). The culture was maintained under the same conditions described above. The light intensity under cool white bulbs was 100 µmol photons m-2 s-1. All experimental cultures were sampled during the logarithmic phase. 2.5. Laboratory experiment We conducted laboratory studies to clarify the relationship between the diel cell cycle and vertical migration of H. akashiwo. Synchronized cells were inoculated to flasks for Experiment 1 or cylinders for Experiment 2, each containing 2 L of F/2 media at a final density of 1 x 104 cells mL-1. These cells were incubated at 20oC under 100 µmol photons m-2 s-1 with a 12:12 h light:dark cycle. Experiments were initiated after 24 h of stabilization. Experiment 1: Diel phasing of the cell cycle progression The amount of DNA in cells was monitored with a flow cytometer every 3 h for 48 h. To confirm that cell division was occurring, cell density was also monitored as described above. Culture flasks containing H. akashiwo were mixed gently and 25 ml were removed for cell cycle analysis and 1 ml for measurement of cell density. Since culture flasks were mixed, we could not evaluate vertical migration in this experiment; however, we analyzed the diel cell cycle timing and cell division. The experiment was conducted in triplicate using three flasks. 2.2. Cell fixation Cells of H. akashiwo were fixed with paraformaldehyde (PFA) at a final concentration of 0.1%. The fixative contains sodium cacodylate (pH 7.4, 0.2 M, final concentration) and sucrose (0.1 M, final concentration) and F-68 (0.1%, final concentration). 2.3. Diel cell cycle analysis and the calculation of the growth rate Fixed cells were washed with deionized water (DW) and ethanol, and suspended with phosphatebuffered saline (PBS, pH 7.5). We confirmed that ethanol treatment removed chlorophyll, which interferes with propidium iodide (PI) fluorescence used in the flow cytometric analysis. Cells treated with RNase (final concentration 20 mg mL-1, Sigma, USA) were incubated at 35oC for 30 min then were stained with 20 µg mL-1 PI (Sigma, USA). Diel cell cycle Experiment 2: Diel phasing of cell cycle progression and vertical migration H. akashiwo cells were incubated using 2 L cylinders (10 cm in diameter 40 cm in height) under the same conditions as Experiment 1. In the preliminary experiment, we found that cells migrated to the surface from CT0 (circadian time, 0 h) at least 31 © 2012 GSTF GSTF International Journal of BioSciences (JBio) Vol.2 No.1, December 2012 until CT9, and to the bottom from CT12 until CT21. We collected samples every 6 h from CT21 without mixing. For the diel cell cycle analysis, samples were taken from the surface during the day and from the bottom during the night. To confirm the diel vertical migration, each surface and bottom sample was measured for in vivo chlorophyll fluorescence using a 10-AU fluorometer (Turner Designs, Sunnyvale, CA, USA). The experiment was completed in triplicate using three cylinders after 24 h of stabilization. day as occurring from CT21 to the following day CT21. The cell density of H. akashiwo increased from 1.6 x 104 cells mL-1 to 4.8 x 104 cells mL-1 after 48 h of incubation (Figure 1. A). The cell density increased from CT21 to CT0 on the first day and from CT18 on the first day to CT6 on the second day. On the second day, cell density increased again from CT18. DNA histograms of H. akashiwo cells are shown in Figure 1. B. A distinct G2/M peak was found in the later dark period (CT18, CT21). These peaks diminished in the light period, indicating cell division. Coefficient of variation (CV) values for these histograms ranged between 6.5% and 17.3% with a mean of 10.8%. G1-phase cells increased from CT15 on the first day, reaching a peak (90%) at CT3 on the second day (Figure 1. C). DNA synthesis began in the early light period; the percentage of cells in the Sphase increased from CT0 on the first day and from CT3 on the second day. These values reached their daily maximum at CT15 and CT18 on the first and second days, respectively. As the percentage of S-phase cells dropped, the percentage of G2+M-phase cells increased. The G2+M-phase cells reached a peak at CT21 on all days. G2+M-phase cells shifted to the G1phase from CT21 to CT6, which coincided with cell division (Figures, 1. A and C). The growth rate based on the diel cell cycle analysis was 0.34 d-1 for the first day and 0.23 d-1 for the second day. The reduced growth rate observed during the second day was probably due to a lower fraction of cells in the S-phase (Figure 1. C). To clarify the relation between vertical migration pattern and cell division in the laboratory, we observed the concentration of chlorophyll fluorescence and the diel cell cycle. High chlorophyll fluorescence from H. akashiwo cells was observed at the surface during the light period (CT3, CT9) and at the bottom during the dark period (CT15, CT21, Figure 2. A). Percentages of fluorescence in the bottom layers increased from 13.4% at CT3 to 20.1% at CT9 on the first day and from 17.9% to 19.7% on the second day, suggesting that a downward migration began during the late light period. The diel cell cycle pattern followed by vertical migration was quite similar to those observed in the previous experiment (Figure 2. B). When the percentage of G1 cells was maximized, the cells formed a patch at the surface (CT3, CT9, Figures, 2. A and B), and the percentage of the G1-phase cells decreased from CT3 (Figure 2. B). The percentage of S-phase cells began to rise from CT9, reaching a peak at CT15 on both days. When the cells started to migrate downward (Figure 2. A), the percentage of S-phase cells also started to increase (Figure 2. B). The percentage of S-phase cells increased from 5.9% at CT3 to 19.2% at CT9 during the first day and from 4.6% at CT3 to 12.1% at CT9 on the second day. When the cells migrated to the bottom, the percentage of S- Experiment 3, Microscopic observation of dividing cells We sampled H. akashiwo cells from the surface at CT3 when they formed a patch on the surface water. This method was used for Experiment 2. The cells were stained with 4’-6-diamidino-2-phenylindole (DAPI) at a final concentration of 1 µg mL-1. Stained cells were observed under UV excitation with an epifluorescence microscope (Axioplan, ZEISS, Germany) at 400Х magnification. Micrographs were also taken with a CCD camera (Qincom fast, Qimaging, Canada). 2.6. Field observations Field observations were performed in Masan Bay (35o 12’ 12.33’’, 128o 35’ 03.95’’), when H. akashiwo formed a dense bloom (ca. 2.4 x 104 cells mL-1) from 20 to 21 June 2007. Vertical water temperature measurements were made with a portable (Model U-10, HORIBA, Japan) at 0, 1, 2, 3, and 4 m. Photosynthetically available radiation was measured vertically with a quantum sensor (Li-250, Li-Cor, Lincoln, NE, USA). Water samples were taken every 4 h from 1900 h on 20 June to 2300 h on 21 June from 0, 1.5 and 3 m with a 3-L Van-Dorn sampler. The water samples were fixed immediately with the fixative mixture as described above. All samples were packed on dry ice and transported to the laboratory, where they were stored in a freezer (-70oC) until the analysis. For the enumeration of H. akashiwo cells, the sample was fixed with Lugol’s solution at a final concentration of 0.5%. To determine the concentration of chlorophyll a (Chl. a) and nutrients, water samples were collected at 1900 h on 20 June and 1500 h on 21 June. A portion (250 ml) of the water sample was filtered with a GF/F filter (Whatman, 47 mm diameter) and the Chl. a concentrations were determined after extraction with 90% acetone (APHA 1995). Each of the nutrient samples (NH4-N, NO3-N+NO2-N and PO4-P) was prepared from 50 ml of filtered water through GF/F filters; the nutrient concentrations were determined following standard methods (APHA 1995). 3.RESULTS 3.1. Laboratory experiments In this study, all laboratory experiments were started at CT21. Therefore, we regarded the first 32 © 2012 GSTF GSTF International Journal of BioSciences (JBio) Vol.2 No.1, December 2012 phase cells maximized and, when the cells remained at the bottom, the percentage of G2+M-phase cells increased from CT15. The percentage of G2+M-phase cells reached a maximum at CT21, and decreased until CT9. This shift of G2+M-phase cells into G1-phase occurred simultaneously with upward migration. To summarize, in the upward migration and shift from M-phase to G1-phase, we observed the nucleus in the cells which were sampled from the surface. Epifluorescence-microscopic observation of cells at CT3 revealed that many cells had a binary nucleus in one cell (Figure 7). It clearly demonstrates that these cells were in the M-phase. values ranged between 13.1% and 16.3% with a mean of 14.5%. The percentage of G1-phase cells decreased from 1900 h on 20 June to 0300 h on 21 June and from 1500 h to 2300 h on 21 June (Figure 5. B). Then, it increased until 1500 h on 21 June. The percentage of the G1-phase cells reached its peak at 1500 h on 21 June. The percentage of S-phase cells increased from 1900 h to 2300 h on 20 June and from 1500 h to 23:00 on 21 June. The percentage of G2+M-phase cells increased from 1900 h on 20 June to 0300 h on 21 June and decreased until 1500 h on 21 June. The percentage of G2+M-phase cells reached a maximum of 14.7% at 2300 h on 20 June. The growth rate based on the diel cell cycle analysis was 0.34 d-1 during the investigated period. This value was higher than the growth rates in the laboratory experiment, probably due to a higher number of S-phase cells (Figure 5. B) in the natural sea water. 3.2. Field observation The concentrations of NH4-N were higher at the surface (3.1 and 6.7 µM) than those at the bottom (1.5 and 2.2 µM, Table 1). The highest concentration of NO3-N +NO2-N was 2.5 µM and the lowest concentration was 2.2 µM (Table 1). There was no trend in its vertical distribution. The concentrations of PO4-P, ranging between 0.6 and 3.1 µM, also showed a similar trend to those of NH4-N. High chl. a concentrations were detected at the surface (306.9 and 466.8 µg L-1, Table 1). Microscopic observation revealed that more than 95% of phytoplankton cells were H. akashiwo. Other phytoplankton taxa present at less than 5% of the total phytoplankton cell concentrations were Cosinodiscus sp. and Thalassiosira decipiens (Bacillariophyceae), Eutreptiella gymnastica (Euglenophyceae), Gymnodinium sp. and Prorocentrum triestinum (Dinophyceae). The temperature at the surface ranged between 22.5 and 25.5oC during the sample period (Figure 4. A). Dawn and dusk occurred, respectively, at 0511 h and 1943 h on 21 June. Light intensity rapidly attenuated with depth from 685.4 µmol photons m-2 s-1 at the surface to 6.8 µmol photons m-2 s-1 at 1m (Figure 4. B) at 1100 h. At that time, the attenuation coefficient was 3.08 m-1, and Z1%, the depth where light was attenuated to 1% of the surface, was 1.50 m. The cell density of H. akashiwo ranged from 0.3 to 13.3 x 104 cells mL-1 (Figure 4. C). H. akashiwo cells dispersed vertically from 1900 h on June 20 when sampling was started. The cells migrated to the surface from 0300 h before dawn and formed a dense patch at the surface during the day to a maximum of 13.3 x 104 cells mL-1. At the same time, the lowest values were recorded at 4 m depth. Most cells were distributed above the Z1% during daytime (Figures, 4. B and C). These cells started to descend around 1500 h, before the sunset. At 1900 h, a patch moving downward was observed at 3 m depth. The DNA histograms of the natural H. akashiwo population are shown in Figure 5. A. Overall, peaks were broad compared to those observed in the strain in the laboratory experiment (Figure 1. B). CV 4.DISCUSSION The rapid appearance of H. akashiwo blooms in coastal environments (Bearon et al. 2006; Handy et al. 2005; Hard et al. 2000; Honjo 1993; Odebrecht and Abreu 1995; Rensel et al. 1989; Smayda 1998; Taylor and Haigh 1993; Yamochi 1987) is due to physical and biological processes. Among them, it is widely accepted that the higher growth rate of H. akashiwo compared to other raphidophytes and dinoflagellates is the reason for the rapid appearances. In addition to rapid growth of H. akashiwo, wind sometimes concentrates H. akashiwo cells near the coast. Thus, in situ growth should be separately evaluated from such wind driven processes in analyzing the bloom development. The cell density of H. akashiwo drastically increased from 0300 h (13.1 x 103 cells mL-1) to 1100 h (133.0 x 103 cells mL-1) (Figure 4. C). From 0700 h (118.0 x 103) to 1100 h (133.0 x 103 cells mL-1), cell density at the surface increased again (Figure 4. C). At the same time, percentage of G2/M-phase decreased from 11.3% to 2.6% (Figure 5. B). G2/M-phase cell numbers at 0700 h and 1100 h were, respectively, 13.3 x 103 and 3.5 x 103 cells mL-1. Therefore, the increase in cell density by cell division between these two times accounted for 9.8 x 103 cells mL-1, while apparent increase was 15.0 x 103 cells mL-1. This means that 65.3% of the apparent increase in cell concentrations was explained by cell division. In contrast, from 0300 h to 0700 h the change in percentage of G2/M-phase was less obvious (14.3% to 11.3%), while increase in cell density was more significant from 13.1 x 103 to 118.0 x 103 cells mL-1. Thus, the cause for the apparent increase in cell density at the surface shifted from vertical migration to the cell division. High cell density of H. akashiwo (Figure 4. C) and chlorophyll concentration (Table 1) in Masan Bay indicated that our investigation was carried out 33 © 2012 GSTF GSTF International Journal of BioSciences (JBio) Vol.2 No.1, December 2012 around the time of at the peak of the bloom. Generally, the growth rate of microalgae becomes low in late exponential and stationary phases, as described by Pan and Cembella (1998). However, we detected positive growth (0.34 day-1) of H. akashiwo in the dense patch, although the rate did not reach a maximum rate previously recorded (1.14 day-1, Yamatogi et al. 2006). This positive growth at the peak of the bloom suggests that losses to grazing and cell dispersion to the adjacent waters may be substantial. Recently, Jeong et al. (2005) pointed out the significance of heterotrophic dinoflagellates as the grazer for H. akashiwo. In analyzing population dynamics, the loss process requires more attention. In the laboratory experiment, G1-phase cells started to increase when they migrated upward (Figures. 2. A and B). Cells undergoing mitosis were found at the surface at CT3 (Figure 3). Moreover, the cell density increased between CT18 and CT6 (Figure 1. A). Indeed, in Masan Bay, H. akashiwo cells migrated upward when G2+M-phased cell decreased before the sunrise (Figures. 4. C and 5. B). In a previous laboratory study, enlarged H. akashiwo cells migrated downward and newly-divided cells started to migrate upwards with cell division and upward migration at CT21 (Kohata and Watanabe 1986). Thus, it is clear that cell division occurs simultaneously with upward migration in H. akashiwo. The overlapping of vertical migration and cell division has been reported for studies of other dinoflagellate species (Table 2). For example, cell division of Prorocentrum micans started at CT2 (Pan and Cembella 1998), while upward migration of this species started at CT22 (Kamykowski 1981). In Lingulodinium polyedrum, cell division started 2 h before dawn in the laboratory (Homma and Hasting 1989). Upward migration was also found to have started at dawn in Baja California (Blasco 1978). In the case of Karenia brevis, cell division appeared to be completed before dawn (Van Dolah and Leighfield 1999; Van Dolah et al. 2008), and upward migration also started before dawn (Van Dolah et al. 2008). It has been demonstrated that the diel cell cycle progression and vertical migration behavior are under the control of circadian rhythm (Ault 2000; Brunelle et al. 2007; Van Dolah and Leighfield 1999; Weiler and Karl 1979). The variation in these timings can be a factor to determine phytoplankton succession. Although rhythms in vertical migration and diel cell cycle seem to be synchronized, it is not known whether there is interaction between the two. There was no difference in the percentage of different cell phase in K. brevis between depths (Van Dolah and Leighfield 1999; Van Dolah et al. 2008). Thus, Van Dolah et al. (2008) concluded that cell cycles of K. brevis occur independently of vertical migration behavior (Van Dolah et al. 2008). However, vertical positioning of this dinoflagellate species was affected by the internal cellular condition (Kamykowski et al. 1998). For H. akashiwo, we observed that vertical migration became less obvious when the culture entered into a stationary phase (data not shown). In the present study, most cells of H. akashiwo were concentrated at the surface; hence, we could not analyze cells in the sub-surface layer. It should be further determined in laboratory experiment. The height of the cylinder used for our vertical migration experiment was no more than 40 cm, while of > 1 m height mesocosms were employed previously to study vertical migration behavior (Dublin et al. 2006; Kamykowski et al. 1998; Kohata and Watanabe 1986; Watanabe et al. 1988). In the vertical migration experiment, most cells accumulated at the surface during the light period, and the thickness of the H. akashiwo cell patches was less than 5 (data not shown). During the dark period, cells concentrated on the bottom of the cylinders. Thus, we believe that vertical migration of H .akashiwo observed in the present study well presented their original behavior. H. akashiwo grow more rapidly (1.14 day-1) than the dinoflagellates such as Karenia brevis (0.69 day-1). Honjo (1993) observed that a cell of H. akashiwo divided up to three times during one night. We showed that the cell cycle progression of H. akashiwo seemed to be synchronized. However, both the laboratory experiments and the field study did not show more than one division per day. High initial cell density (1 x 104 cells mL-1) in the laboratory experiments is one possible explanation for the lower growth rate (< one division per day). It is of interest whether cells can divide more than one time during a night without accumulation of carbon by photosynthesis. This is a topic requiring further investigation. One of the potentially important ecological advantages of vertical migration behavior is nutrient uptake from deeper layers during the night. However, our data did not support this idea; concentrations of NO3-N and PO4-P were higher in the surface layer than the bottom (Table 1). This leads to a tentative conclusion that in the bay, vertical migration behavior does not give such an advantage for H. akashiwo over other competitive phytoplankton species. Another explanation for the lower nutrient concentrations at the bottom of the bay is a result of nutrient uptake by H. akashiwo cells during night time. Since our investigation was carried out at the peak of the bloom, it is possible that these nutrients had been already depleted. Unfortunately, we do not have conclusive date to decide this issue. It is certain, however, that nutrient dynamics is one of the important environmental factors for the development of H. akashiwo population in the bay. In conclusion, we demonstrated a diel cell cycle of H. akashiwo for both a cultured strain and natural populations. Upward migration and cell 34 © 2012 GSTF GSTF International Journal of BioSciences (JBio) Vol.2 No.1, December 2012 division appeared to occur simultaneously. The increase in cell density at the surface in Masan Bay was partly due to cell division, although bloom had already developed. To more accurately analyze the bloom developmental process, the present study emphasizes that population growth should be separately evaluated from horizontal advection and vertical migration. Since the growth rate of this harmful alga is high compared to that of other dinoflagellates, the cell cycle behavior of H. akashiwo may differ from that of dinoflagellates. Our study shows that diel cell cycle analysis is a useful tool to clarify the bloom development process, and the method will provides us with important new insights. 8 229-239 Handy SM, Coyne KJ and Portune KJ 2005 Evaluating vertical migration behavior of harmful raphidophytes in the Delaware Inland Bays utilizing quantitative real-time PCR. Aquatic Microbial Ecology 40 121-132 Hard JJ, Connell L and Hershberger WK 2000 Genetic variation in mortality of chinook salmon during a bloom of the marine alga Heterosigma akashiwo. Journal of Fish Biology 56 1387-1397 Hill AE 1991 Vertical migration in tidal currents. Marine Ecology Progress Series 75 39-54 Homma K and Hasting JW 1989 The S phase is discrete and is controlled by the circadian clock in the marine dinoflagellate Gonyaulax polyedra. Experimental Cell Research 182 635-644 Acknowledgements This work was supported by the Korea Institute of Ocean Science and Technology Research Fund (#PE98747). 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Nippon Suisan Gakkaishi 72 160- Taylor FJR and Horner RA 1994 Red tides and other problems with 168 harmful algal blooms in Pacific Northwest coastal waters. Proceedings of the Symposium on the Marine Yamochi S 1987 Mechanisms for outbreak of Heterosigma akashiwo Environment, Canadian Technical Reports of Fisheries red tide in Osaka Bay, Japan. In Proceedings of the 1st International Symposium on Red tides pp 253-256 and Aquatic Science, pp 175-186 Taylor FJR and Haigh R 1993 The ecology of fish-killing blooms of Yamochi S and Abe T 1984 Mechanisms to initiate a Heterosigma the chloromonad flagellate Heterosigma in the Strait of akashiwo red tide in Osaka Bay II. Diel vertical Georgia and adjacent waters. In: TJ Smayda and Y migration. Marine Biology 83 255-261 36 © 2012 GSTF GSTF International Journal of BioSciences (JBio) Vol.2 No.1, December 2012 Figure legends Figure 1 (A) The growth curve of H. akashiwo strain (HYM06HA) based on the cell counting. (B) Cell cycle progression of H. akashiwo strain HYM06HA. Histograms of DNA fluorescence of cell determined by flow cytometry. Experiment was carried out in triplicate, and results from one of the representative flask are shown. (C) Ratio of cell phase (%) which was analyzed of each cell phase from the laboratory experiment. All experiments were conducted over 48 h with 3 h intervals. ■: G1, ●: G2+M, ▲: S, CT: circadian time. Figure 2 (A) The daily fluctuation of percentages of cell migration based on the Chl. a concentration. Each sample was collected at the top and bottom over 48 h with 6 h intervals. (B) Ratio of cell phase (%) from the laboratory experiment over 48 h. The cells of H. akashiwo collected from the patch were analyzed for their diel cell cycle at 6 h intervals. ······ : G1, ―: G2+M, ---: S, □: Dark period, ●: Light period, CT: circadian time. Figure 3 Binary nucleus in one cell of H. akashiwo strain HYM06HA, which is stained with DAPI. Chlorophyll showed red auto-fluorescence with the DAPI-specific filter set. These cells in two figures were collected at CT2 from the top of the cylinder. Figure 4 Changes in the vertical profiles of water temperature (oC, panel A), light intensity (µmol photons m-2 s-1, panel C) and vertical distribution of H. akashiwo cells (x 103 cells mL-1) in Masan Bay from 20 to 21 Jun 2007. Figure 5 (A) Cell cycle progression of H. akashiwo population in Masan Bay. Histograms of DNA fluorescence of cell determined by flow cytometry are shown. (B) Ratio of cell phase (%) from the field experiment over 28 h. The daily fluctuation of each cell phase was analyzed at 4 h intervals. ■: G1, ○: S, ▲: G2+M. Figure 1 37 © 2012 GSTF GSTF International Journal of BioSciences (JBio) Vol.2 No.1, December 2012 Figure Figure 3 38 © 2012 GSTF GSTF International Journal of BioSciences (JBio) Vol.2 No.1, December 2012 Figure 4 39 © 2012 GSTF GSTF International Journal of BioSciences (JBio) Vol.2 No.1, December 2012 Figure 5 Table 1. The vertical concentrations of nutrients (NH4, NO3+NO2, PO4) (µM) and Chl. a (µg L-1) in Masan Bay on 20 and 21 June 2007. Date 20 June 2007 Time Depth 19:00 0m 1.5m 3m 21 June 2007 15:00 0m 1.5m 3m NH4 (µM) 6.7 NO3+NO2 (µM) 2.5 3.1 1.5 2.3 2.4 3.1 2.4 0.0 2.2 2.2 2.3 40 PO4 (µM) Chl.a (µg L-1) 1.8 466.8 1.3 1.0 68.9 111.8 3.1 306.9 0.6 0.9 18.9 59.1 © 2012 GSTF GSTF International Journal of BioSciences (JBio) Vol.2 No.1, December 2012 Table 2. Beginning times (circadian time, CT or local time) of cell division (CD) and upward migration (VM) for various species of flagellates. Species Location L:D Beginning times cycle CD VM Method Reference Dinophyceae Karenia brevis Lingulodinium polyedrum Laboratory 16:08 CT16CT22 Gulf of 16:00- Mexico 21:00 Laboratory 12:12 Laboratory 12:12 Prorocentrum minimum 1999 Van Dolah et al. 2008 FL Komykowski et al. 1998 FCM Homma and Hasting 1989 LM Blasco1978 FCM Pan and Cembella 1998 FL Kamykowski 1981 FCM Pan and Cembella 1998 CT2 LM Watanabe et al. 1982 CT22 CT0 California Van Dolah and Leighfield FCM CT2 Baja Prorocentrum micans FCM Laboratory 12:12 CT2 Laboratory 12:12 Laboratory 12:12 Laboratory 12:12 Laboratory 12:12 CT21 CT21 LM Kohata and Watanabe 1986 Laboratory 12:12 CT21 CT21 FCM This study Masan Bay 14:10 CT21 CT21 FCM This study CT22 CT4 Raphidopyceae Heterosigma akasniwo *FCM: flow cytometry *FL: fluorometry (in vivo fluorescence) *LM: light microscopy 41 © 2012 GSTF