Using cell cycle analysis to estimate in situ growth rate of the dinoflagellate dinophysis acuminata: Drawbacks of the DNA quantification method

Abstract

In an attempt to use cell cycle analysis to estimate in situ gross growth rate of the dinoflagellate Dinophysis acuminata, epifluorescence microscopy in combination with an image analysis system was used to measure the relative DNA content of DAPI stained D. acuminata nuclei. To be able to estimate growth rate with this method, the time it takes for a cell to synthesise a second DNA copy and complete mitosis (the duration of the terminal event in the cell cycle) must be known or measurable. The duration of the terminal event is normally estimated graphically from diurnal variations in the phase fraction curves. No diurnal variation in the phase fractions was detected in this study, and consequently no reliable estimate of the duration of the terminal event could be obtained. The main draw, back was the difficulty in delineating the S phase in DNA histograms based on only a few hundred cells. No clear S phase maximum could be obtained during our 48 h study. The presence of doublenucleated cells and a constantly high percentage (23 to 43 %) of cells with double genomes (G2 + M phase cells) suggests, under the assumption that no cells can arrest in the G2 or M phase, that the population was actively dividing, but not clearly in phase with a diurnal cycle. Chang and Carpenter (1991) previously estimated the duration of the terminal event (the duration of the S + G2 + M phases) in this species to be 11 to 13 h. A 12 h duration of the terminal event in this study would yield specific growth rates of 0.69 to 0.75 d-1. We conclude that the number of cells that can be measured using epifluorescence microscopy (a few hundred per sample) is too low to allow detection of a low degree of synchronisation, especially with regard to the S phase. Estimations of in situ growth rate of poorly synchronised populations of phytoplankton using the cell cycle technique will require DNA measurements on several thousand cells per sample, e.g. using flow cytometry or automated image cytometry.