Hydrobiologia 238: 139-147, 1992.
T. Berman, H. J. Gons & L. R. Mur (eds), The Daily Growth Cycle of Phytoplankton.
© 1992 Kluwer Academic Publishers. Printed in Belgium.
139
Variations in biochemical parameters of Heterocapsa sp. and
Olisthodiscus luteus grown in 12:12 light:dark cycles
I. Cell cycle and nucleic acid composition
E. Berdalet, M. Latasa & M. Estrada
Institut de Cibncies del Mar, CSIC, Pg. Nacional s/n 08039 Barcelona, Spain
Key words: Heterocapsa, Olisthodiscus, RNA/DNA, cell cycle
Abstract
The division cycle of two phytoplankton species, Olisthodiscus luteus and Heterocapsa sp. was studied
in relation to a 12:12 light:dark cycle. Batch cultures in exponential phase were sampled every three hours
during 48 hours. Cell number, cellular volume and DNA and RNA concentrations were measured.
Microscopic observations of the nuclei of Heterocapsa sp. were also performed. In both species, cell
division took place in the dark. In Heterocapsa sp., DNA and RNA showed a similar diel variability
pattern, with synthesis starting at the end of the light period, previously to mitosis and cytokinesis. In
0. luteus. Major RNA synthesis occurred during darkness, and DNA was produced almost continuously. Both species presented different values and diel rhythmicity on the RNA/DNA ratios.
Introduction
Measurements of RNA/DNA ratios and DNA
concentrations have been used as biochemical indicators of physiological state and biomass of
planktonic marine organisms (Bulow, 1987;
Bmstedt & Skjoldal, 1980; Clemmesen, 1988)
including phytoplankton (Dortch et al., 1983;
1985). Many of these studies have concerned the
diagnosis of nutrient limitation of microalgal
growth (Flynn, 1990). Nevertheless, both DNA
and RNA are important molecular compounds
directly implicated in the cell growth and division
processes, and they vary in relationship with these
processes. Thus, understanding the magnitude of
this physiological variation is of fundamental importance for the use of RNA and DNA as biochemical indicators (Eppley, 1981).
In their natural environment, phytoplankton
cells are exposed to a regularity and rhythmicity
of the light supply which often results in the syn-
chronization of many cellular events with the
photocycle (Chisholm, 1981). Rhythms in nutrient assimilation, photosynthetic activity and cell
division have been studied in natural populations
and laboratory cultures (Eppley, 1981). Rhythmicity in cellular RNA (Waltz et al., 1983) and
DNA (Karentz, 1983) concentrations has also
been described, but few works treat simultaneously cell division cycle and RNA and DNA
synthesis (Ricketts, 1977; and see revision in
Puiseux-Dao, 1981). The present work deals with
the variability of the concentration of RNA and
DNA, in relationship to the cell division cycle in
batch cultures of two phytoplankton species: the
dinoflagellate Heterocapsa sp. Stein and the
raphidophyte Olisthodiscus luteus N. Carter.
Parallel studies of pigment concentrations were
performed on part of the same cultures (one for
each species); the results are reported in Latasa
et al., 1992.
140
Materials and methods
Olisthodiscus luteus N. Carter, Raphidophyceae
(according to Chr6tiennot-Dinet, 1990), was provided by the Plymouth Marine Laboratory, UK;
Heterocapsa sp. Stein (sensu Morrill & Loeblich,
1981), Dinophyceae, was isolated from Barcelona
Harbour waters. Two batch cultures of each species were started in 4 1 flasks containing 3 1 of f/
2 media (Guillard, 1975) without silicate, at a
constant temperature of 18 + 1 C and under a
12:12 h light-dark period with a light intensity of
150 molm - 2 s- . Inocula were taken from
adapted and exponentially growing cultures. Due
to unknown reasons one of the replicates of Heterocapsa sp. did not grow and could not be used
for the experiment. Daily cell counts were done
with a 3.1 ml settling chamber. In the exponential
phase, cultures were sampled during 48 hours at
1.5 h intervals for Coulter Counter measurements
and microscopic observations of the cells and at
3 h intervals for the rest of parameters (see below).
Prior to sampling, flasks were carefully swirled to
insure even distribution of cells.
Cellular volume, total cell volume (TCV, volume of cells per ml of culture) and cell number
were estimated in vivo using a Multisizer Coulter
Counter, with a 140 m aperture tube, and a 32
channel particle-size analyzer. Cell numbers were
additionally estimated by means of the inverted
microscope technique (Utermohl, 1958) using
3.1 ml settling chambers. For 0. luteus similar cell
number estimates were obtained with the inverted
microscope and the Coulter Counter; the calculations were based on the Coulter Counter data.
However, in the case of Heterocapsa sp., good
correlations between both cell number estimates
were only obtained during the first 24 hours. This
occurred because the increase in cell density during the course of the experiment, surpassed the
acceptable % coincidence level in the Coulter
Counter, and produced an underestimation of the
particle number and total particle volume, although did not affect the volume:particle ratio
(other experiments, data not shown). Thus, the
calculations of growth rate and nucleic acid content per cell are referred to microscopic cell num-
ber counts, but cellular volume was obtained by
the quotient between Total Cell Volume (TCV)
and cell number measured by the Coulter
Counter.
Observation of the Acridine Orange stained
nuclei of cells settled in a 3.1 ml chamber, were
carried out with an inverted epifluorescence microscope. The cells were treated with an Acridine
Orange-formaldehyde mixture. 1 ml of a solution
consisting of 200 g ml- ' acridine orange in concentrated 37% calcium carbonate buffered formaldehyde was added to 19 ml of sample, Coats &
Heinbokel, 1982.
Growth rates were calculated from the regression of ln(Nj) on time, ti, where Ni was the cell
number at 16:00 hours of the i = 0, 1 and 2 days
of the experiment. The growth constant, j (t- 1)
was the slope of the regression line and was converted to divisions per day by the equation: k
(div.day- ) = ,u/ln 2 (Guillard, 1973).
Samples for RNA and DNA determination
were vacuum filtered (< 10 mm Hg) through precombusted (350 C, 24 h) Whatman GF/F filters
which were subsequently stored in liquid nitrogen. Nucleic acids were analyzed by a new double staining fluorescence technique (Berdalet &
Dortch, 1991). RNA/DNA ratios were expressed
on a nitrogen weight basis; assuming a composition of 16.84% nitrogen by weight for DNA and
16.12% for RNA (Dortch etal., 1983).
The experimental sampling and analytical variability of the DNA and RNA determination was
estimated in an independent experiment, using
replicate samples taken from experimentally
growing cultures of 0. luteus and Heterocapsasp..
Mean values + standard error of the mean for
DNA were respectively, 19.77 + 1.70 (n = 6) g
DNA 1- and 67.19 + 3.96 (n = 4) g DNA 1;
and the corresponding figures for RNA were
114.75 + 2.81 (n= 6) ig RNA 1- and 354 + 12.02
(n = 4) g RNA 1- 1, respectively.
As an approximation, supported by the lack of
gas vacuoles in both species, we considered that
for each of them, cell volume was proportional to
biomass, as measured in terms of carbon (Hitchcock, 1982, Kohata & Watanabe, 1988). Thus,
the nucleic acid content referred to total cell vol-
141
multaneous increase in cellular volume and total
3 cell volume/ml culture,
cell volume (TCV, /Am
Fig. la), started shortly after the onset of the light
period and lasted until the beginning of darkness.
The microscopic observation of cells and nuclei revealed different phases, which are shown in
Fig. 2. Cells originating from a cellular division
(interphasic cells) have a small and compact nuResults
cleus located in the epitheca (a). Progressively,
Heterocapsa sp.
the nucleus grows, elongates and moves towards
the central zone (cingulum) (b, c). Finally, nuclear
During the course of the experiment the growth
division and cytokinesis takes place in an oblique
rate was 1.06 divisions per day (as calculated
plane (d, e). Stages b and c can be included in the
from data of Fig. la). Cell division processes took
mitotic events and the last phases (d, e) correplace during the dark peri od and beginning of the
spond to cytokinetic processes. The timing of the
light period, as revealed by the increase in cell
mitotic and cytokinetic events is shown in Fig. 3.
number (Fig. la) and the decrease in cellular volThe percentage of cells undergoing mitotic (b and
ume (Fig. lb). Cellular g rowth, measured as sic) or cytokinetic stages (d) was computed each
1.5 h interval. The first changes in the nuclei
(Fig.
2a-c) started in the second half of the light
,,
period. The maximum proportion of cells exhibiting a large, elongated and central nucleus was
E
25
reached during the first half of the dark period.
V
Cytokinesis took place mostly during the second
0
Po
20
half of darkness and at the beginning of the light
0
period. Observed cells undergoing cytokinesis
n 15
(Fig. 3) were few. This could indicate that the last
E
steps of mitosis and cytokinesis occurred much
3C
faster than the first mitotic events.
10
U
Both DNA and RNA showed a similar vari3
6/////DX.
ability
pattern, with major synthesis starting to50
12
24
36 hour48
wards the end of the light period and continuing
ume was assumed to express contribution to the
biomass of the cell.
To show more clearly any rhythmic trends, the
data were smoothed using;sliding means for every
two consecutive data poiJnts.
0N
sented L/D rhythmicity (Fig. 4b) with an amplitude of about 1/6th of the ratio. Minimum values
were found during the middle of the dark period
and maximum values close to the light onset. The
75
V
E
0
0
E
To
L_
u
a
U
hours
Fig. 1. Diel changes in cell number (a) and cellular volume (b)
in Heterocapsa sp. (shaded areas indicate the dark period).
b
c
d
e
Fig. 2. Cellular phases observed by microscopy in Heterocapsa sp.: (a) interphasic cells; (b) nucleus increases in volume
and (c) moves to the cingulum; (d and e) chromosome separation and cytokinesis in an oblique plane.
142
7A
/U
0
60
50
z
0
m 40
40
01
::3
a)
OL
&R 30
20
10
R
.8
hours
Fig. 3. Diel changes in the mitotic and cytokinetic processes
in Heterocapsa sp.
6,51
9:~
0
tY
DNA synthesis period preceded the start of cell
division (Figs. la,b and 4c). Nucleic acid content
related to TCV presented also diel rhythmicities,
with maximum ratios at the end of darkness and
minimum values in the middle of the light period
(not shown).
According to our measurements in this experiment, the estimated DNA content per cell in an
haploid nucleus would be about 3.5 pg cell - . The
maximum DNA content per cell, corresponding
to the end of the DNA duplication was 7 pg-
·
A
50
1
40
0
cell - and 9 pg cell- 1 on the first and the second
night, respectively.
V
0
ct
20
CL.
0.
10
Olisthodiscus luteus
During the experiment, the two replicate cultures
presented a growth rate of 0.96 divisions per day
(as estimated from data of Fig. 5a). Cell division
and cellular growth were strongly phased with the
L:D cycle (Figs. 5a-c). Cell division took place
during the night as revealed by the pattern of
increase in cell concentration (Fig. 5a). During
the day, the increase in TCV (Fig. 5b) revealed
that cellular growth occurred. The average cellular volume (Fig. 5c) showed a marked diel cycle,
with a decrease corresponding to cell division
10
0
12
24
36
n
48
hours
Fig. 4. Diel changes in nucleic acids in Heterocapsa sp..
(a) total DNA and RNA concentration; (b) RNA/DNA ratios; (c) cellular DNA and RNA content.
processes and an increase related to cellular
growth.
Although nuclei were well stained by Acridine
Orange, it was not possible to observe mitotic
143
during the time of the experiment (Fig. 6a). The
increase in the ratio DNA/TCV (Fig. 6c) during
the night, when TCV remained constant (Fig. 5b),
revealed more clearly the major synthesis period.
During the day, when the TCV increased, the
ratio dropped. DNA content per cell showed diel
rhythmicity with maximum ratios during the night
14
E
12
0)
'U
10
.0
8
13 6
C
4
(7 pg cell- 1) and minimum values during the day
(4pg cell -) (not shown). This indicated that
.5
U
2
-0
12
24
36
N
S 30
hours
4,
b
0
25
E
.E 20
2
15
P
10
5
0
12
24
36
hours
4
A
DNA synthesis preceded mitosis.
RNA varied in a different way than DNA
(Fig. 6b). Its concentration increased during the
first night, decreased during the following 24
hours, and starting in the last hours of the second
night, showed a strong increase which continued
into the first hours of the second light period.
The ratio RNA/DNA varied markedly during
the sampling period, but was not in phase with
cell division, due to the pattern of RNA variation
(Fig. 6c). The first RNA/DNA maximum occurred in the middle of the dark period, and the
second one after the light onset.
Discussion
2,2
2
U 1.8
E 1,6
1,4
1,2
1
hours
J flask 1|
2
IO flask 21
Fig. 5. Diel changes in cell number (a), TCV = total cell volume (b) and cellular volume (c) in 0. luteus. Data of the individual flasks.
events by microscopy, because the cytoplasm of
0. luteus collapsed when cells were fixed.
The DNA concentration per unit volume of
culture increased during the light and dark period
The determinations of DNA, RNA and cell volume and the microscopic observations (in the case
of Heterocapsa sp.) suggest that the populations
of both species were in phase with the L:D cycle,
i.e. undergoing particular cell cycle stages at the
same time over the L:D period.
Traditionally, the cell cycle has been divided in
5 discrete sequential intervals: G1, S, G2, M and
C. G1 and G2 are, respectively, the gaps separating DNA synthesis (S phase) and cell division
(mitosis, M, and cytokinesis, C). In our experimental conditions, cell division (M and C) of Heterocapsa sp. took place during the night (Figs. la
and 3). Cellular growth, estimated by the increase
in cellular volume (Fig. lb) occurred during the
day. DNA synthesis (S) started in the last hours
of the light period (Figs. 4a & 4c), simultaneously
to the nucleus displacement from the anterior part
of the cell towards the cellular center, which corresponds to the first steps of mitosis (Figs. 2 & 3).
Therefore, the cells must expend all the light time
144
on the G1 phase (from the end of the cell division
until to the start of DNA synthesis), and G2 must
be very short or non existent.
This pattern is comparable to that observed in
H. triquetra by Chang & Carpenter (1988), which
computed the DNA content by microfluorimetry.
A similar coupling of DNA synthesis, mitosis and
cytokinesis has also been found in other dinoflagellates, such as Heterocapsa pygmaea,
Gymnodinium nelsonfi, Amphidinium carterae and
Prorocentrum triestinum by Karentz (1983). This
author considered that a short or absent G2 would
insure the occurrence of DNA replication prior to
mitosis and cytokinesis, and thus it would avoid
the expenditure of energy for the maintenance of
an extra complement of nuclear material. Nevertheless, interspecific and intraspecific variations
in the pattern of DNA synthesis and cell division
coupling were observed within the different dinoflagellates (Karentz, 1983).
A decrease in cell number accompanied by a
decrease in the DNA and RNA content of the
population occurred after the time of maximum
cell concentration within each L:D cycle. Similar
decreases in cell number at periodic intervals over
24-h cycles have been reported in field observations and dinoflagellates in culture (Nelson &
Brand, 1979; Pollingher & Zemel, 1981; Karentz,
1983). Karentz suggested that some particular
stages of the cell cycle may show high susceptibility to cell mortality. An increased cell mortality could perhaps be due to the mechanical stress
of swirling the cultures prior to sampling. It is not
likely, that sexual processes (conjugation) were
implied in a decrease of cell number, because the
corresponding cellular forms were not observed
by microscopy. The decrease in the nucleic acids
could be due to catabolic reactions (in the case of
RNA) and/or the lysis of cells with release of
RNA and DNA, which are not retained by the
filters.
a
70
50
t
a)
=0
30-
:
12
,,
20
0
,
I
:/,,4,::,//i::,4hu,
24
36
hours
4;8
4
C
3, 5
3
I
N2, 5
2
1, 5
0
6
12
- _r
, ,
24
36
hours
91///
418
I
d
5,5
5
E4,5
z4
;"%
3,5
3
2,5
'0
12
24
36
'
hours
4
flask 1
flask
Fig. 6. Diel changes in nucleic acids in 0. luteus. (a) total
DNA concentration; (b) total RNA concentration; (c) DNA
content referred to cellular volume; (d) RNA/DNA ratios.
Data of the individual flasks.
145
Table 1.
Phytoplankton species
RNA/DNA
Reference
Amphidinium carterae
(dinoflagellate)
Thalassiosira weissflogii
(diatom)
Platymonas striata
9.3-13
Ricketts, 1977
Other organisms
RNA/DNA
Reference
Several bacteria species
1-6
Dortch et al., 1983
1-9
Dortch et al., 1983
1-14
Dortch et al., 1983
(several sources)
Clemmesen, 1988
Raae et al., 1988
Clarke et al., 1989
Fish larvae
Gadus morhua
Sepia officinalis
Pecten maximus
feminine gonad
male gonad
3.8-11
3-12
0-50
3-7
0.5-0
Robbins et al., 1990
Natural samples
RNA/DNA
Reference
North Pacific Ocean
Dabob Bay
North Pacific Ocean
(dissolved RNA and DNA)
Coastal seawaters near Oahu, Hawaii
(dissolved RNA and DNA)
1-18
2-15
4.1-11.5
Takahashi et al., 1974
Dortch et al., 1985
Karl & Bailiff, 1989
4.5-10.9
Karl & Bailiff, 1989
In the case of 0. luteus, it was not possible to
follow mitotic changes by microscopy. Nevertheless, if we assume, based on the results for Heterocapsa sp., that changes in cellular volume were
good indicators of the mitotic events and growth
processes, the circadian rhythmicity of cellular
volume would indicate that mitosis took place
during darkness and cellular growth during the
light period.
The progressive increase of the DNA concentration in the cultures during the light and dark
periods (Fig. 6a), suggested a continuous DNA
synthesis. Nevertheless, DNA concentration referred to total cell volume (Fig. 6c), implies that
the major DNA synthesis took place mostly at
night, continuing shortly during the day (Fig. 6a).
We suggest that the cellular cycle was probably
very similar to that of Heterocapsasp., with an S
phase preceding mitosis, and a very short or nonexistent G 2 period.
The RNA/DNA ratios have been considered
as a biochemical indicator of the growth rate,
which is determined in part by environmental
conditions (Dortch et al., 1983; Flynn, 1990). In
the eukaryotes, DNA is assumed to be almost
constant, varying only by a factor of 2, except in
the case of polyploidy (Thomas & Carr, 1985).
The maximum variation in the DNA content of
the cells is the doubling of the chromosomes prior
to division. RNA is supposed to be a function of
the rate of protein synthesis, which should increase with increasing rate (Dortch et al., 1983).
Some of the available data (obtained by different techniques) are shown on Table 1. Most
works illustrate the RNA/DNA variation with
the different growth rates resulting, in general,
from non-limiting or limiting nutritional conditions. The RNA/DNA ratios obtained in our
study fall within the range of phytoplankton presented in Table 1. It is interesting to note that the
two studied species, growing under identical optimal environmental conditions, with a very sim-
146
ilar growth rate, presented different values of
RNA/DNA ratios. In Heterocapsasp. the RNA/
DNA ratio varied from 4.7 to 6.6, and in 0. luteus
from 1.4 to 3.2. The diel oscillation pattern and
the amplitude of the oscillation around the mean
value were also different: about a 20% in the
per a la Recerca i la Innovaci6 Tecnol6gica,
CIRIT, de la Generalitat de Catalunya.
dinoflagellate and a 40% in the raphidophyte.
Platymonas striata (Prasinophyceae), growing at
Bamstedt, U. & H. R. Skjoldal, 1980. RNA concentration of
zooplankton: relationship with size and growth. Limnol.
Oceanogr. 25: 304-316.
Berdalet, E. & Q. F. Dortch, in press. New double staining
technique for measuring RNA and DNA in marine phytoplankton. Mar. Ecol. Prog. Ser. 73: 295-395.
Bulow, F. J., 1987. RNA-DNA ratios as indicators of growth
in fish: a review. In: R. C. Summerfelt & G. E. Hall (eds),
The age and growth of fish, The Iowa State Univ. Press,
Ames, Iowa, USA: 45-64.
Chang, J. & E. J. Carpenter, 1988. Species-specific phytoplankton growth rates via diel DNA synthesis cycles. II.
DNA quantification and model verification in the dinoflagellate Heterocapsa triquetra. Mar. Ecol. Prog. Ser. 44: 287296.
Chisholm, S. W., 1981. Temporal patterns of cell division in
unicellular algae. In: T. Platt (ed.), Physiological basis of
phytoplankton ecology. Can. Bull. Fish. aquat. Sci. 210:
150-210.
Chr6tiennot-Dinet, M-J., 1990. Atlas du phytoplancton marin.
Vol. 3, p. 120. Ed. CNRS, Paris.
Clarke, A., P. G. Rodhouse, L. H. Holmes & P. L. Pascoe,
1989. Growth rate and nucleic acid ratio in cultured cuttlefish Sepia officinalis (Mollusca: Cephalopoda). J. Exp.
mar. Biol. Ecol. 133: 229-240.
Clemmesen, C., 1988. A RNA and DNA fluorescence technique to evaluate the nutritional condition of individual
marine fish larvae. Meeresforch. 32: 134-143.
Coats, D. W. & J. F. Heinboken, 1982. A study of reproduction and other life cycle phenomena in planktonic protists
using an acridine orange fluorescence technique. Mar. Biol.
67: 71-79.
Dortch, Q. F., T. L. Roberts, J. R. Clayton, Jr. & S. I. Ahmed,
1983. RNA/DNA ratios and DNA concentrations as indicators of growth rate and biomass in planktonic marine
organisms. Mar. Ecol. Prog. Ser. 13: 61-71.
Dortch, Q. F., J. R. Clayton, Jr., S. S. Thoresen & S. I.
Ahmed, 1985. Nitrogen storage and the use of biochemical
indices to asses nitrogen deficiency and growth rate in natural plankton populations. J. mar. Res. 43: 437-464.
Eppley, R. W., 1981. Relationship between nutrient assimilation and growth in phytoplankton with a brief review of
estimates of growth rate in the ocean. In: T. Platt (ed.),
Physiological basis of phytoplankton ecology, Can. Bull.
Fish. aquat. Sci. 210: 251-263.
Flynn, K. J., 1990. The determination of nitrogen status in
microalgae. Mar. Ecol. Prog. Ser. 61: 297-307.
Guillard, R. R. L., 1973. Division rates. In: J. R. Stein (ed.),
Handbook of phycological methods. I. Culture methods
0.85 doublings per day on a 14:10 L:D cycle, at
optimal light and nutrient conditions (Ricketts,
1977), presented RNA/DNA values between 9.3
to 13, with an oscillation of 17% (Ricketts, 1977).
The different diel fluctuations of the RNA/DNA
ratios, are a consequence of the particular variability patterns of the RNA and DNA concentrations in each species during the light:dark cycle.
Our results illustrate that there is large interspecific variability of the RNA/DNA ratios for
species growing in very similar environmental
conditions. Presumably, different phytoplankton
species may display different responses to the environmental changes (light and/or nutrient supply) that affect the RNA/DNA ratio. This could
explain in part the dispersion found in the correlations between growth rates and RNA/DNA ratios reported in the literature (see for example,
Dortch etal., 1983; Raae etal., 1988; Clarke
et al., 1989).
In this work we report that important variations in the value of RNA/DNA ratios may arise
from species-specific differences and from fluctuations associated to cell cycle. Understanding
these sources of variability is important in order
to evaluate the usefulness of RNA/DNA ratios as
a biochemical indicator of phytoplankton physiology.
Acknowledgements
The authors are grateful to E. Saiz for the comments on the laboratory work. We also thank Dr
P. J. Wangersky who reviewed the manuscript.
This research was supported by FPI grants from
the Ministerio de Educaci6n y Ciencia de Espafla
and funds from Programa Fronts MAR 88-0252,
CYCIT, CSIC., and the Comissi6 Internacional
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