THE ECOLOGICAL UNDERSTANDINGS FOR GROWTH, CELL CYCLE, AND VERTICAL MIGRATION OF HETEROSIGMA AKASHIWO (RAPHIDOPHYCEAE)

Abstract

To understand the blooming process of Heterosigma akashiwo, I investigated its growth, cell cycle, and vertical migration. Additionally, I developed the cell cycle analysis technique using the tyramide signal amplification-fluorescence in situ hybridazation (TSA-FISH) for the field application. The optimum probe and formamide concentrations for TSA-FISH probe were revealed to be 0.5 ng μL-1 and 40%. The specificity of this probe was confirmed by testing non-target phytoplankton including Chattonella sp. which has the most similar sequence information, and the probe did not hybridize to tested phytoplanktons. I could successfully identify G1- and G2-phase cells and the diel cell cycle of H. akashiwo not only in the laboratory but also in the field after TSA-FISH. Thus, TSA-FISH for H. akashiwo in natural coastal waters could be used for understanding their bloom mechanism using flow cytometer. Then, I designed an equation to estimate cell division more than one time per day based on the cell cycle and then calculated in situ growth rates. At this study period, in situ growth rates of H. akashiwo were range from 0.59 to 1.13 d-1. I conclude that this newly derived equation improves the ability to accurately estimate bloom formation by fast cell division. I investigated the growth patterns of 3 different H. akashiwo strains under various physiological conditions. All 3 strains survived in a wide range of salinity from 10 to 40 psu. The NFHTS-AK-1 and CCMP 452 survived at 5 psu, however HYM06HA did not survive. The growth patterns under various N:P ratios at 6, 10, 14, 18, and 22 and selenium concentrations from 10-3 to 100 nM were significantly different depending on the Korean strains and USA strain. The effections of iron concentrations were tested from 0.0044 to 4.4 M and the growth pattern were not significantly different. The growth rate of Korean strains, NFHTS-AK-1 and HYM06HA, were enhanced by N:P ratio and micronutrient, selenium and iron. The growth rate of CCMP 452 was enhanced by micronutrient, iron. To understand the the diel cell cycle progression and vertical migration of H. akahsiwo, I investigated this phenomenon in the laboratory and natural environment. In laboratory experiments, cell division of H. akashiwo began approximately 3 h before the beginning of the light period. Cells accumulated at the surface during the light period, and migrated to the bottom during the dark period. Epifluorescence microscopy revealed that cells collected from the surface were in mitosis phase at 3 h after the beginning of the light period. In the field experiments, cells divided from 2 h before sunrise to 4 h after sunrise and cells began to migrate to the surface before sunrise when these cells started to divide. Both experiments clearly showed that vertical migration to the surface accompanies cell division. To clarity the relationship between cell division and vertical migration, I arrested the cell cycle of H. akashiwo with inhibitor. However, the vertical migration of cells kept. The vertical migration pattern was maintained under continuous light and dark conditions. And upward migration started when the light-on signal started 3 hours earlier than in the control. I confirmed that the vertical migration of H. akashiwo is regulated by circadian rhythm and that the rhythm is triggered by a light-on signal. The diel vertical migration was both controlled by circadian rhythm and initiated by a light-on signal. After onset of the circadian rhythm, the direction of migration could be decided by phototaxis. The results of the present study suggested specific physiological cue for the blooming mechanism of H. akashiwo with developed methodology and new insight about the mechanisms of vertical migration cue.