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Vol.
23:
251-255, 1985
MARINE ECOLOGY
-
PROGRESS SERIES
Mar. Ecol. Prog. Ser.
I
Published May
31
Application of a single-cell isolation technique to
studies of carbon assimilation by the subtropical
silicoflagellate
Dictyocha perlaevis
*
Satoru Taguchi
&
Edward
A.
Laws
Hawaii Institute of Marine Biology
&
Department
of
Oceanography, University
of
Hawaii at Manoa,
1000
Pope Road, Honolulu,
Hawaii
96822.
USA
ABSTRACT:
A
single-cell isolation technique was used to study the assimilation of
14C0,
by natural
populations of the silicoflagellate
Dictyocha perlaevis.
The method uses a concentrated natural sample
and a high activity of radioisotope. The technique facil~tates
physiological
studies of certain species
which cannot be conveniently studied in the laboratory due to difficulty in maintaining laboratory
cultures. Data obtained from a subtropical embayment are used to illustrate the application of the
method, and include in particular a study of carbon partitioning patterns into major polymers over the
course of a die1 cycle.
INTRODUCTION
Silicoflagellates are marine chrysophytes bearing a
single flagellum and a star-shaped silica skeleton com-
posed of tubular elements. They have golden-brown
chromatophores and are photosynthetic. Silicoflagel-
lates are found in most parts of the world's oceans, and
have been reported as one of the most important foods
for warm-water salps (e.g. Gemeinhardt 1934). How-
ever, the nutritional value of silicoflagellates has never
been studied due to the difficulty of raising cultures in
the laboratory. For example, van Valkenburg
&
Norris
(1970)
have reported the only successful effort to grow
Dictyocha fibula
in culture, and most information
about the morphological characteristics of this species
and its growth rate as a function of temperature and
salinity were obtained as a result of that single study.
Subsequent efforts to culture
D.
fibula
have been un-
successful.
For physiological studies the difficulties in culturing
silicoflagellates in the laboratory can be partially over-
come by conducting experiments in the field, but only
if a satisfactory method exists for separating silico-
flagellates from the other seston. Rivkin
&
Seliger
(1981) have previously described a single cell isolation
technique which involves coarse screening of a sample
Hawaii Institute
of
Marine Biology, Contribution No.
699
O
Inter-Research/Printed in
F.
R.
Germany
followed by isolation of single cells using a micro-
pipette and dissecting microscope. Use of this tech-
nique on natural populations of silicoflagellates could
greatly expand our ability to study the physiology of
this important class of phytoplankton. We report here
results of the use of the single-cell isolation technique
to measure inorganic carbon uptake and incorporation
into major polymers by natural populations of the
silicoflagellate
Dictyocha perlaevis
Frenquelli.
MATERIALS
AND
METHODS
Dictyocha perlaevis
was isolated on
14
Sep 1982
from a water sample collected in Kaneohe Bay, Hawaii
at 157O47.2'
W,
21" 25.4'
N.
A
35
pn
mesh plankton net
with a 30 cm diameter opening was towed vertically
from 2 m above the bottom to the surface. The total
volume of water filtered was about 3001. The sample
was prescreened through a 183 Km mesh net to remove
large zooplankton. The following
2
experiments were
then conducted immediately. First, in an approach
which we will refer to as the Rivkin-Seliger method,
the water collected from
1
tow was partitioned into 2
light and
1
dark bottles (125 m1 capacity). Each sub-
sample was inoculated with 50pCi of NaH14C03 and
incubated for 24 h at a constant temperature of 24°C
and under an irradiance of 400 pEinst m-2s-1 provided
252
Mar. Ecol.
Prog.
Ser.
23: 251-255, 1985
on a 12L: 12D cycle by a bank of cool-white fluorescent
lamps. The incubations were begun at approximately
0800 local time, and the
L:D
cycle was timed so as to
begin the dark period 12 h later. One m1 aliquots were
taken from each bottle to determine the total activity of
'"C. At the end of the incubations the contents of each
bottle were concentrated with a 20 Km mesh strainer
and washed carefully with 14C-free filtered (0.4 pm)
seawater.
D.
perlaevis
cells were then isolated using a
micropipette and dissecting microscope, and washed
twice with '"C-free filtered seawater. Finally the cells
were transfered into scintillation vials for subsequent
fractionation.
In a parallel experiment,
Dictyocha
perlaevis
cells
from a second tow were pipetted into 2 light and 1 dark
Petri dishes containing approximately 5 m1 of filtered
seawater collected from the same station. Each dish
was inoculated with
5
kCi of NaH14C0, and incubated
in the same manner as the 125 m1 bottles. At the end of
the incubation only swimming cells were pipetted out
under the dissecting microscope. The cells were
washed twice with 14C-free filtered seawater and
transfered into a scintillation vial. Triplicate vials con-
tained either
1,
2, 4,
8,
16, 32 or 64 cells per vial. We
will refer to this second approach in which only swim-
ming cells were pipetted from the Petri dishes as the
modified Rivkin-Seliger method. All vials then
received an addition of 10 m1 of scintillant (Amersham
ACS) and were stored in the dark prior to counting on a
Searle Analytic liquid scintillation counter. Quench
corrections were made by the external standard ratio
method.
Chlorophyll
a
determinations were made on dupli-
cate samples containing at least 40 cells which had
been previously isolated, serially transfered through
filtered seawater, and collected on Whatman
GF/C
glass fiber filters. Chlorophyll
a
was extracted in 100
%
acetone and measured on a Turner fluorometer using
the procedures recommended by Holm-Hansen et al.
(1965).
On 2 other occasions a water sample from the same
station was concentrated and incubated in two 125 m1
light bottles under conditions similar to those used in
the first experiment.
Dicfyocha
perlaevis
cells were
isolated from aliquots taken from the
2
bottles after 6,
12 and
24
h.
After the
D.
perlaevis
cells had been
isolated and placed in scintillation vials, a differential
extraction technique first employed by Morris et al.
(1974) was used to partition the recently assimilated
carbon between protein, polysaccharide, lipid, and
small molecular weight intermediate compounds. The
procedure was similar to that outlined in DiTullio
&
Laws (1983). Three m1 of a 2:
1
(v:v) chloroform
:
methanol mixture were added to each vial, and the
vials vortexed for
1
min to extract the lipids and small
molecular weight intermediate compounds. The con-
tents of each vial were then filtered through a GF/C
glass fiber filter, and the residue washed 3 times with
3 m1 of the chloroform
:
methanol mixture to remove
any remaining lipid or intermediate compounds. The
residue was resuspended in 3 m1 of
5
%
trichloroacetic
acid (TCA) and heated at
85
"C for 30 min to solubilize
polysaccharides, and nucleic acids and to precipitate
protein. After acid hydrolysis the slurry was filtered
through another GF/C filter and rinsed with
2
m1 of
cold (0°C)
5
%
TCA. The filtrate obtained from the
chloroform: methanol extraction was transfered into a
separatory flask and 3 m1 of 0.88% KC1 was added.
-
1
wash
3x
I
WPLE
F1
LTER
wash ~ith Fig.
1.
Flow diagram
of
solvent
Polysaccharides
2
mls cold extraction procedure to separate
plus
hbcleic
flltrate (o'c)s%
TCA
protein, polysaccharide, lipids,
Acids and low molecular weight inter-
residue mediate compounds. Chloroform
2:l
Ch:bleCH
.
with
3
mls
*
then vortex
ch
:MEGi
filtrate
-
3
mls of
Y
=
Ch; methanol
=
MeOH; low-
for one min. lresidue
07
layer
P
Protein
rl
molecular-weight intermediates
=
LMWI
Add
3
mls of
0.88%
KC1;
then allo~i to
separate
(
.\.
30
min)
5%
TCA;
heat at
SS'C
for
30
mln.
residue
f
\f
hieCH
filtrate
layer
1
Add
3
mls
of
I:l?l&H:H20
hleOli
:H20
-
*
layer
\
Taguchi
&
Laws: Application of a single-cell isolation technique
After separation was complete, the
2
layers were trans-
fered into separate scintillation vials. The chloroform
layer was then transfered back to a separatory flask,
and
3
m1 of a
1
:
1
(v: v) methanol :water mixture was
added for a final wash and the immiscible solvents
separated. The water-methanol layer was then com-
bined with the original methanol layer. The fractiona-
tion technique thus separated the cell material into
TCA-insoluble compounds (protein), TCA-soluble
compounds (polysaccharide plus nucleic acids),
chloroform soluble compounds (lipid) and methanol-
water soluble compounds (low molecular weight inter-
mediates). The volume of each of the liquid fractions
was reduced by evaporating the samples at room tem-
perature for
16
h before addition of the scintillant. A
flow diagram of the separation procedure is indicated
in Fig.
1.
If
6
samples are processed at a time,
24
samples can be processed in an
8
h work-day. Scintil-
lation counting procedures were identical to those
used in the previous experiment.
RESULTS
Table
1
provides a comparison of results obtained
with the original and modified Rivkin-Seliger techni-
ques.
A
regression analysis of
DPM
vial-' against cells
vial-' gave virtually identical results, and in both
cases the relationship was highly linear (r
=
0.999).
The Y-intercepts of the regression equations were not
significantly different from the radioactivity of the dark
bottles.
Fig.
2
and Table
2
give a good indication of the
feasibility of using the carbon partitioning technique
Table
1.
Results of linear regression analysis (cells vial-'
vs
DPM vial-') of the original method of Rivkin
&
Seliger
(1981)
and the modified Rivkin-Seliger method
Parameters Method
Original Present
Y-intercept
30.9 31.2
Slope
15.9 16.2
r
0.999 0.999
Table
2.
Comparison of sum of
4
fractions and unfractionated
total DPM
Cell numbers Sum of
4
Unfractionated Ratio of
vial-' fractions total sum to total
8 1,769 1,698 1.04
16 4,038 4,129 0.98
3 2 7.962 8.216 0.97
64 15.301 14.998 1.02
CELLS
PER
VIAL
Fig.
2.
Djctyocha
perlaevis.
Rate of
I4CO2
incorporation into
protein
(o),
polysaccharide
(q),
lipids
(A),
and low-molecular-
weight metabolites
(0)
as a function of the number of isolated
cells vial-'. Protein:
Y
=
-32
+
66X
(r
=
0.998);
polysac-
charide:
Y
=
-28
t
43X
(r
=
0.995);
lipids.
Y
=
50
+
42X
(r
=
0.996);
low-molecular-weight metabolltes:
Y
=
92
+
88X
(r
=
0.995)
-
EXP
M
'
AUGUST
3,
1983
HOURS
Fig.
3.
Dictyocha
perlaevis.
Rate of
14C0,
incorporation into
protein
(O),
polysaccharidc~
(U),
lipids
(A)
and low-molecular-
weight metabolites
(0)
as
a
function of time of
12L: 12D
light
cycle incubation. White and black portions on X-axis repre-
sent light and dark periods. Each value is mean
t
l
standard
deviation
254
Mar. Ecol. Prog. Ser.
23: 251-255, 1985
in conjunction with the Rivkin-Seliger isolation
method. Due to the low radioactivity compared to
blanks and a high variability of radioactivity between
samples with 4 or fewer cells per vial, only data from
vials with 8 or more cells were fitted to a linear regres-
sion line. All regression slopes in Fig. 2 were highly
significant (p
<
0.01), and the sum of the 14C02 incor-
poration rates into the 4 fractions was not significantly
different from the rate determined from unfractionated
controls (Table 2).
In a final experiment on 3 Aug 1983 14C02 incorpora-
rion rates for
Dictyochea perlaevis
were measured into
major polymers using triplicate vials containing 10
cells each. The results are shown in Fig. 3 and Table 3.
Total uptake rates were almost identical during the
first and second 6 h of the photoperiod. Respiration and
exc~etion losses resulted in a slightly negative incorpo-
ration rate during the dark period.
Table
3.
Percent protein, polysaccharides, lipids, and low-
molecular-weight metabolites of total
I4CO,
uptake at the end
of light and dark period
Protein Poly- Lipids Low-molecu-
saccha- lar-weight
rides metabolites
Light
31.1
43.0 5.8 20.1
Dark
30.7 38.0
8.1
23.1
DISCUSSION
The Rivkin-Seliger method was originally applied to
species of coastal dinoflagellates, but was later proven
to work for presumably more fragile oceanic dino-
flagellates (Rivkin et al. 1984). The results reported
here confirm the validity of the cell isolation technique
with one of the most fragile marine phytoplankton
species. In particular, the original Rivkin-Seliger
method, in which no effort was made to discriminate
between motile and non-motile cells, and the modified
Rivkin-Seliger method, in which only motile cells were
pipetted into scintillation vials, gave virtually identical
results (Table
1).
The good agreement between the
original and modified Rivkin-Seliger methods is
noteworthy, since the cells were collected in net tows,
and the possibility exists that at least some cells were
stressed in the process of collection. However, there
was no apparent difference between the amount of
14C02 assimilated by
Dictyocha perlaevis
cells which
were clearly motile at the end of the 24 h incubation,
and those cells which showed no evidence of motility.
The photosynthetic rate of
Dictyocha perlaevis
cells
on
14
Sep 1982 was 2.0gC (g Chl a)-'h-' during the
photoperiod, a figure which is well within the range of
0.8 to 6.2 cited by Malone (1980) for netplankton light-
saturated photosynthetic rates. The estimated biomass
and 14C02 uptake rate of
D. perlaeviswas
608 pg Chl
a
1-I and 1.23 ngC
1-I
h-', respectively. This rep-
resented 53 and 36
%
of the corresponding netplankton
(35
pm)
measurements, respectively. Thus the photo-
synthetic rates of
D. perlaevis
cells appear to be com-
parable to that of other netplankton in Kaneohe Bay.
We conclude that all of our data are consistent with the
assumption that the Rivkin-Seliger cell isolation
technique provides a satisfactory means for studying
the physiology of these fragile silicoflagellates in the
field.
Since Morris et al. (1974) combined the standard
14C02 method (Strickland
&
Parsons 1972) with the
carbon partitioning method of Roberts et al. (1963) to
estimate the incorporation rate of 14C02 into major
polymers, the carbon partitioning technique has
become a promising method for studying the physio-
logy of phytoplankton (DiTullio
&
Laws 1983, Redalje
&
Laws 1983, Terry et al. 1983, Cuhel et al. 1984).
Recently there has been some controversy regarding
the incorporation of 14C0, into lipids by polar species
(Smith
&
Morris 1980, Barlow
&
Henry 1982, Li
&
Platt
1982). Differences in species composition have been
suggested as a possible explanation for the discrep-
ancy of carbon assimilation into lipid (Li
&
Platt 1982).
However, only general floristic descriptions are avail-
able with which to check this hypothesis. The feasibil-
ity of using the Rivkin-Seliger cell isolation technique
to study carbon partitioning by individual species is
therefore of interest. The results shown in Fig. 2 and
Table
2
indicate that I4CO2 incorporation into major
polymers can be precisely determined for
Dictyocha
perlaevis
with the Rivkin-Seliger technique when as
few as 8 cells are placed into triplicate vials after an
incubation of 6 h or more.
In the field experiment summarized in Fig. 3, the fact
that total uptake rates were very similar during the first
6
h and last 6 h of the photoperiod suggests that the
results were probably not biased by bottle confinement
artifacts. The insignificant rate of protein synthesis
during the dark period presumably reflects the lack of
carbon storage products at the end of the photoperiod
(Terry et al. 1983, Cuhel et al. 1984). The percentage of
14C02 incorporated into protein suggests that the cells
were growing at a rate equal to about 45
%
of the
maximum possible rate at the prevailing temperature
(DiTullio
&
Laws 1983).
We conclude that the Rivkin-Seliger technique can
be successfully applied to species as fragile as silico-
flagellates, and that the carbon partitioning approach
of Morris et al. (1974) can be combined with the Riv-
kin-Seliger cell isolation method to facilitate phy-
siological studies of individuals species of algae in the
Taguchi
&
Laws: Application of a single-cell isolation techniques 255
field. This fact may have profound implications in
physiological ecology if species are found that have
drastically differently carbon assimilation patterns to
other species (e.g. Smith
&
Morris
1980).
Knowledge
in
this area in conjunction with microscopic analysis of
the species composition present at an experimental
site would prove invaluable in deciphering data that
are both temporally and spatially distinct.
LITERATURE CITED
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G.,
Henry,
H.
L. (1982). Patterns of carbon assimila-
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A.
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K.
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This paper was submitted to the editor; it was accepted for printing on March 23, 1985