NAGATA, TOSHI. The microflagellate-picoplankton food ... - ASLO

Limnol. Oceanogr., 33(4, part l), 1988, 504-517
Q 1988, by the American Society of Limnology and Oceanography, Inc.
The microflagellate-picoplankton food linkage in the
water column of Lake Biwa
Toshi Nagata
Otsu Hydrobiological Station, Kyoto University, Shimosakamoto,
Otsu, 520-01 Japan
Abstract
Seasonal variations of abundance and biomass of heterotrophic microflagellates (HMF), bacteria,
and unicellular chroococcoid cyanobacteria (UCB) at a pelagic site (water depth, -72 m) in the
north basin of Lake Biwa were measured during May to November 1984. The cell densities of
HMF, bacteria, and UCB were 102-1 03, lo”, and 102-lo5 cells ml- I, and the biomass estimates
were < 1-7, 1 O-70, and < l-20 pg C liter - I. Active phagocytosis of UCB by HMF was indicated
from microscopic observation of HMF food vacuoles; when UCB density was high, up to 52% of
HMF cells contained one or more UCB cells. Incubation of size-fractionated epilimnetic waters
indicated that bacterial net production rates (5-25 pg C liter-’ d-m’) were usually almost equal to
rates of consumption of bacteria by HMF populations. Estimates of gross growth efficiencies of
HMF fed on natural picoplankton were 1 l-53% in terms of carbon. HMF may be a significant
link transferring picoplankton energy to macrozooplankters which cannot collect picoplankton.
Lack of information on the fate of HMF makes it difficult, however, to directly address the “sink
vs. link” argument about the microbial loop in Lake Biwa.
It has been hypothesized that heterotro-
phic microflagellates (HMF) are major con-
sumers of planktonic bacteria (Fenchel
1982~; Sieburth and Davis 1982; Wright
and Coffin 1984; Glide 1986) and picophy-
toplankton (Johnson et al. 1982) in lakes
and seas. Since picoplankton can be signif-
icant components of biomass and produc-
tion (Fuhrman and Azam 1982; Larsson and
Hagstrom 1982; Stockner and Antia 1986),
HMF may play important functional roles
in the cycling of matter in these environ-
ments. Two opposing hypotheses exist con-
cerning these roles (Sherr and Sherr 1984;
Porter et al. 1985). Some investigators have
stressed that zooplankters, for which pico-
planktonic particles are too small to collect,
but which can collect HMF, indirectly USC
picoplankton resources via the HMF-pi-
coplankton food chain. Others believe that
---
Acknowkdgments
Contribution 3 18 from Otsu Hydrobiological Sta-
tion, Kyoto University (foreign language series). This
work was partly supported by a grant for Scientific
Research (6 1790227) from the Ministry of Education,
Science and Culture of Japan.
I thank Y. Tezuka for advice and encouragement
throughout this study. Thanks are also due to other
researchers of Otsu Hydrobiological Station for dis-
cussions, to A. Kawabata and T. Ueda for assistance
in water sampling, and to I). Scavia, L. Carlough, and
T. Fenchcl for suggestions on the manuscript.
through the ingestion and digestion of pi-
coplankton by HMF, substantial amounts
of the organic carbon, nitrogen, and phos-
phorus contained in picoplankton are re-
leased to the environment as regenerated
minerals. Most of the data supporting these
ideas have been obtained from experiments
with cultured HMF strains. Little quanti-
tative information is available about bio-
mass, grazing rate, and production rate of
HMF and their prey in natural environ-
ments (Fenchel 1982~; Sherr et al. 1984).
My objective here was to examine the
seasonal and vertical variations of the cell
abundance and biomass of HMF, bacteria,
and unicellular chroococcoid cyanobacteria
(UCB) in a water column of the north basin
of Lake Biwa, Japan. Grazing rates of the
HMF on UCB were estimated from direct
counts of UCB in food vacuoles of HMF
and retention times reported in the litera-
ture. Grazing of bacteria by HMF was as-
sessed by bottle incubation experiments with
size-fractionated lake waters.
Study site
Lake Biwa is near 35’N, 136”E. Its major
water basin is the north one, of which sur-
face area is 6 18 km2 and mean water depth
is 46.5 m. Recent trophic state (mesotro-
phic: Tezuka 1984), bacterial level (Nagata
1984, 1986a, 1987), and picophytoplank-
504

tonic composition and biomass (Nagata
19863) at the same site have been described.
UCB are the predominant picophytoplank-
ton in the study site, and the picoplankton
contributes 45% of the total Chl a in late
summer (Nagata 1986b).
Materials and methods
Sample collection in the field and enu-
meration of plankton -The sampling site
(water depth, -72 m) was in a pelagic area
of the north basin of the lake. Water samples
were collected with a 3-liter Van Dorn water
sampler. On board, 50 or 100 ml of the
sample from each depth was fixed with 2%
Formalin (final concn) for enumeration of
bacteria and UCB, and another 100 ml of
the sample from each depth was fixed with
glutaraldehyde (electron microscopic grade,
final 1%) for counting HMF. Samples were
kept cool (-4°C) in the dark until they were
processed (within 2 weeks).
Bacteria were counted by the acridine or-
ange (AO) direct count method (Hobbie et
al. 1977) with slight modifications; l-2 ml
of the sample was mixed with 8-10 ml of
particle-free deionized water (PFDW), and
this diluted sample was filtered through a
0.2-pm pore-size Nuclepore filter (stained
with Sudan black B; diam, 24 mm). Im-
mediately after filtration, 0.0 1% AO was
added until it covered the whole filter sur-
face. After 3 min, the AO was drawn
through. About 2 ml of PFDW was filtered
to remove excess dye. Thereafter, the wet
filter was placed on a clear slide glass and
mounted in low-fluorescence immersion oil.
For counting UCB, slides were made with
the same procedure but without AO stain.
Microscope specimens for HMF were made
with fluorescein isothiocyanate (FITC)
(Sherr and Sherr 1983). A lo-50-ml aliquot
of the sample was filtered through a l-pm
pore-size Nuclepore filter (stained with Su-
dan black B). Then FITC (O.Ol%, dissolved
in 0.067 M phosphate buffer of pH 7.2) was
added, and the sample was stained for 1
min. After rinsing the filter with buffer, it
was mounted on a glass slide. For each sam-
ple, duplicate filters were used for bacteria
and HMF and one filter was used for UCB.
The ranges of the duplicate counts were usu-
ally < 15% of the mean values.
Microbial food chain 505
All the filters were observed in a darkened
room under a Nikon epifluorescence micro-
scope (1,250 X) equipped with a standard
B-excitation system (50-W halogen lamp,
IF 4 lo-485 excitation filter, DM 505 di-
chroic mirror, and 5 15 W barrier filter). Bac-
teria framed within an eyepiece grid (40 X
40 pm) were counted in each of lo-20 ran-
dom fields per filter for a total of 200-400
cells. The number of attached cells was
counted for each sample. Due to high vari-
ability, however, it was impossible to reli-
ably estimate attached cell densities. For
each sample, 20 randomly selected cells were
sized with an ocular micrometer, and the
cell volume was calculated by assuming the
shape of bacteria to be spherical or cylin-
drical with hemispherical ends. The volume
of cells so small that they could be recog-
nized only as dots were assumed to be 0.02
pm3. Preliminary examinations showed that
the biovolume estimates (50 cells measured
for each sample) with an ocular micrometer
agree well with those from photographic en-
largements; the ratio (mean cell volume by
a micrometer) : (mean cell volume by pho-
tographs) was 0.8-l .5 (mean 1.2, SD = 0.24,
n = 5). Discrimination of bacteria and UCB
was usually possible in AO samples, be-
cause UCB fluoresced bright orange and
most bacteria fluoresced green. Moreover,
UCB in Lake Biwa were larger and more
swollen than bacteria (Nagata 1986a, b).
Counting UCB was done in the same man-
ner as bacteria; I counted a total of 60-200
cells per sample. I judged orange-fluorescing
unicellular rods and cocci as UCB (Nagata
19863).
For counting HMF, an 80- x 80-pm eye-
piece grid was used. I counted 100 frames
per filter and > 100 cells per sample. I judged
green-fluorescing, flagellated cells to be
HMF. They were discernible from red- or
orange-autofluorescing, pigmented cells
(Nagata 1986b). Sizing of HMF with an ocu-
lar micrometer was done for - 100 cells
randomly selected for each sample. Cell vol-
ume was calculated assuming the shape of
HMF to be spherical or ellipsoidal. The
number of HMF cells that contained at least
one UCB cell in food vacuoles was noted,
and the frequency of HMF containing UCB
was estimated for each sample. Since some

506 Nagata
UCB cells were retained on the 1 -pm pore-
size filter, if an HMF cell landed on top of
a UCB, the HMF might be judged as con-
taining the UCB. Although this background
could not be decided rigorously for each of
my samples because the retention efficiency
of UCB on the l-pm pore-size filter was not
determined for each sample, a rough esti-
mate showed that the background percent-
age was 6, 12, and 18% for the UCB density
of 1, 2, and 3 x 1 O5 cells ml-l. I did not
observe “aggregation” of UCB around
HMF.
The biomass of bacteria, UCB, and HMF
was estimated by multiplying the cell vol-
ume by carbon-to-volume conversion fac-
tors ( 106 fg C pm-3 for bacteria and UCB:
Nagata 1986a; 7 1 fg C pm-3 for HMF: Fen-
chel and Finlay 1983).
Bottle incubation experiments-Water
samples were collected at the site of the field
study during warm months in 1984 and
1985. Samples collected at a depth of 5 or
10 m (epilimnion) with a 3-liter capacity
Van Dorn water sampler were immediately
treated on board with sterile apparatus and
filters as follows. The samples were gravity
filtered through a 25-pm mesh-size screen,
and a portion of this filtrate was further fil-
tered through 1 -pm pore-size Nuclepore fil-
ters with gravity or gentle negative pressure
(< 100 mm of Hg). To reduce clogging, I
filtered no more than 200-300 ml of sample
through one filter (47-mm diam). About 800
ml of each filtrate (25-pm prefiltered water
or 1 -pm prefiltered water) was poured into
a l-liter capacity glass bottle with a glass
plug (previously acid washed and combust-
ed at 450°C for 2 h), and the bottle was then
incubated at in situ water temperature
(+2”C) in the dark. From each bottle, sub-
samples (30-50 ml) were withdrawn with a
sterile pipette or decanted at intervals of 2-
8 h for 24-30 h. Subsamples were fixed with
1% glutaraldehyde, and cell density and bio-
mass of bacteria and HMF were measured
as described above. From 30 to 40 cells of
bacteria and about 100 cells of HMF were
sized for each subsample. For each subsam-
ple, frequency of dividing cells (FDC) of
bacteria was measured according to Hag-
strijm et al. (1979).
The time-course change of bacterial den-
sity in bottled water was analyzed, assuming
that bacterial growth in the 1 -pm prefiltered
sample was exponential and the fraction of
bacteria eliminated per unit of time in the
bottle with grazers is constant [dnldt = rn,
dN/dt = (r - g)N, where dn/dt and dN/dt
are the changing rates (cells liter- * h-l) of
bacterial density in the l-pm prefiltered
sample (n, cells liter- I) and the 25-pm pre-
filtered sample (N, cells liter- I), respective-
ly, and r and g the instantaneous rates of
growth and grazing (h-l)]. The significance
of the effect of differential filtration on bac-
terial dynamics was tested statistically by
comparing the slopes of the regression equa-
tions with Student’s t-test [the regression of
log(bacteria1 density) vs. time was used].
Production. rate, P(cells liter-l d- l), and
grazing rate, G(cells liter-’ d-l), were cal-
culated as follows:
and
P = fNJexp(24 X r’) - l]
GE’-rt
-N,[exp(24 X r’) - l]
r’
where r is the slope of the 1 -pm prefiltered
sample, r’ the slope of the 25-pm prefiltered
sample, and No the ccl1 number at time zero
in the 25-pm prefiltered sample.
The most basic assumptions of the model
are that growth rates of bacteria in lake water
and in the l- and 25-pm prefiltered water
are equal and that grazing occurs only in the
25-pm prefiltered water. These assumptions
will be influenced by accumulation of sub-
strate on the bottle surface and the enhance-
ment of bacterial activity associated with
the surface (Ferguson et al. 1984) enrich-
ment of dissolved organic matter through
filtration (Ferguson et al. 1984; Fuhrman
and Bell 1985) elimination of particulate
organic matter (Glide 1986), and passage of
grazers through a l-pm filter (Cynar et al.
1985). To assess changes of the physiolog-
ical state of bacteria in bottled waters during
the incubation periods, I determined the
time-course changes of FDC and bacterial
mean cell volume. A significant increase in
FDC during the incubation was occasion-
ally found in both the l-pm and the 25-pm

prefiltered water. In most cases (12 of 16),
however, no significant change of FDC was
detected (P > 0.05; two-tailed t-test with
arcsine-transformed FDC values). I found
no systematic difference in FDC between
the 25-pm and the l-pm prcfiltered water.
As to mean cell volume, the regression anal-
yses did not show any significant changes
during the incubation period (P > 0.05, two-
tailed t-test with log-transformed cell vol-
ume). Therefore I assumed that bottle and
filtration effects were minimal. I also count-
ed HMF passing through the l-pm pore-
size filter and found that ~5% of HMF
passed.
Growth parameters of HMF were ob-
tained from the regression of log(HMF
number) vs. time and log(mean cell volume)
vs. time.
Determination of chlorophyll a concentra-
tion -Chl a concentration was measured by
the fluorometric method (Strickland and
Parsons 1972). Volumes of 20-100 ml of
water were filtered through Whatman GF/ C
filters for the field study or through mem-
brane filters (0.2-pm pore size) for the in-
cubation experiments. I used finer meshed
filters for the incubation experiments in or-
der to efficiently recover the Chl a in the
1 -pm prefiltered samples. Chl a was extract-
ed with 90% acetone and then measured
with a spectrofluorometer calibrated with
pure Chl a.
Results
Seasonal variation-Water temperature
of the surface layer increased from 15” (May)
to 3 1°C (20 August) and then decreased to
14” (November) (Fig. 1). Marked stratifi-
cation was established between June and
September with a thermocline between 10
and 20 m. Partial mixing began in October.
Hypolimnetic water temperature was 6”-7°C
during the study period. Chl a concentration
in the upper layer (O-l 0 m) changed dras-
tically between 0.7 and 42.8 pg liter-‘. Peak
values were found in May (Chl a concn, 42.8
pg liter-‘), June (6.9 hg liter-l), and July to
early August (15.4 pg liter-l).
HMF abundance was in the range of 0.8-
5.1 X 1 O3 cells ml-’ in the upper 20-m layer
and 0.1-1.9 x lo3 cells ml-l below 20 m
(Fig. 2). Highest HMF densities were found
Microbial food chain 507
70 M ‘J ’ J ‘A ‘S ’ 0 ’ N
Fig. 1. Seasonal and vertical variation of water
temperature (“C) in the north basin of Lake Biwa.
at 10 and 15 m-the bottom of the epilim-
nion and the metalimnion during summer
stratification. In contrast with HMF, bac-
terial vertical distribution was, in general,
more closely related to water temperature:
higher in the epilimnion than in the meta-
and hypolimnion (Fig. 2). Epilimnetic bac-
terial density ranged between 1.6 and 7.0 x
lo6 cells ml-‘; the meta- and hypolimnion
range was 1.2-3.4 x 10” cells ml-‘. The
highest density was found at 5 and 10 m
during mid-July to early August when a co-
lonial diatom, Fragilaria crotonensis, was
abundant in these layers. UCB could be de-
tected in the whole water column, but the
density below 25 m was very low (0.8-l 6 x
1 O3 cells ml-‘). In the upper 20-m layer, a
drastic increase from 3 x lo4 cells ml-l to
30 x 1 O4 cells ml- I, followed by a decrease
to 3 x lo4 cells ml-‘, was found between
August and November (Fig. 2).
Size and biomass of HMF, bacteria, and
UCB- Detailed taxonomic study on HMF
was not done in the present study. Colony-
forming HMF or HMF associated with par-
ticles were not common, except in late July
and early August at 10 m, where many HMF
attached to frustulcs of F. crotonensis and
detrital particles. Mean cell volumes of HMF
fluctuated seasonally in the range of 9-50
pm3, but no clear seasonal trend was found
(data not shown). Mean cell volumes were,
however, significantly different in different
water layers. I compared the mean cell vol-
umes of HMF in the samples collected at
O-l 0, 10-20, and 20-70 m and found that
cell volume increased with depth (Mann-
Whitney U-test, two-tailed, P < 0.05). At
all depths, the majority (> 70%) of the cells
had an equivalent spherical diameter of 2-

508 Nagata
E
r
it
30
40
50
60
70
0
10
20
30
40
50
G 60
v I 70 i
J ’ J ’ Ai
I- r
M’ J
HMF Bacteria UCB 20
103celis ml-’ IO6 cells ml-’ 1 O4 cells ml”
m 5- 6- 25 - 30
4 -5 5-6 20 - 25
m 3-4 4-5 15-20 40
a 2-3 3-4 10 - 15
0
-2 -3 - 10
,
J
0
,
A
Bacteria
,,
S 0
Fig. 2. Seasonal and vertical variations of the cell abundance of heterotrophic microflagellates (HMF),
bacteria, and unicellular chroococcoid cyanobacteria (UCB) in the north basin of Lake Biwa. Samples were
collected for depths of 0, 2.5, 5, 10, 15, and 20 m at intervals of 7-10 d, and for depths of 30 (or 40), 50 (or
60), and 70 m at intervals of l-4 weeks. For the upper 20-m layers, total numbers of samples analyzed for
HMF, bacteria, and UCB were 129, 134, and 72, and for the layers >20 m they were 47,48, and 22. UCB were
counted after 6 August.
50
60
,:
N

4 pm. For the deeper water layers, however,
the distribution was skewed to larger size
categories.
Because of the high variability within
depth strata, I estimated the attached bac-
terial contribution by pooling the data for
each depth during the whole investigation
period. The percentage of attached bacteria
by number was 0.8, 1.8, and 3.6% for O-l 0,
1 O-30, and 30-70 m. Therefore, it is evident
that unattached bacteria were numerically
predominant in the study site. Mean cell
volume of bacteria was calculated for each
date and each water layer (O-l 0, 1 O-20, and
20-70 m) because the sizing of 20 cells per
sample was not large enough to treat the
data of each sample separately. The mean
cell volume pooled for each date and each
layer (n = 40-80) varied in the range of
0.07-o. 16 pm3. I could not find a clear sea-
sonal trend. The average values of the mean
cell volumes for each layer were 0.10 1 pm3
(SD = 0.019, n = 23), 0.099 pm3 (SD =
0.018, n = 17), and0.121 pm3 (SD = 0.021,
n = 16) for O-l 0, 1 O-20, and 20-70 m. The
average cell volume of the deepest layer was
larger than that of the other two layers
(Mann-Whitney U-test, two-tailed, P <
0.005). These mean values were used to cal-
culate the bacterial biomass for each layer.
I did not measure the size of UCB in the
present study. However, I did observe that
UCB below 15 m were larger and fluoresced
more brightly than those in the upper layers
during 13 August to 20 September. After
partial mixing began, larger forms were
found less frequently in the whole water col-
umn. This pattern was similar to the results
of a previous study (Nagata 1986b). In that
study, the measured mean cell volume of
UCB in 1985 was 0.6 1 pm” for the samples
without larger forms and 2.77 pm3 for the
samples in which larger cells were abundant
(Nagata 19863). I used these values for cal-
culating biomass of UCB in this study.
The biomass of HMF was a tenth to a
twentieth of bacterial biomass, and the bio-
mass of UCB was usually less than a half to
a third of bacterial biomass. HMF biomass
was higher in the middle layer than in the
upper layer during midsummer (Fig. 3).
Conversely bacterial biomass was higher in
the upper layer than in the middle layer
Microbial food chain 509
during the same period. For both the upper
and the middle layers, coupled oscillation
of the biomass of bacteria with that of HMF
was not found. In the lower layers, the bio-
mass estimates of bacteria and HMF were
usually lower than those in the upper and
the middle layers, and they were relatively
stable throughout the investigation. Bio-
masses of UCB in the upper and middle
layers were nearly equal.
The frequency of HMF containing UCB -
I did not observe any HMF containing red-
fluorescing (Chl a-rich: see Waterbury et al.
1979; Murphy and Haugen 1985) eucary-
otic cells in their food vacuoles, although
HMF containing orange-fluorescing (phy-
coerythrin-rich: Waterbury et al. 1979;
Murphy and Haugen 198 5) UCB were often
found. This fact suggests that herbivory of
HMF on eucaryotic phytoplankton was not
prevalent in Lake Biwa.
The percentage of HMF containing UCB
was < 1% until August (data not shown).
Afterward, the percentage increased. At
maximum, 52% of HMF contained UCB in
their food vacuoles (Fig. 4). In most cases,
one HMF cell contained one UCB cell. Al-
though the plots scatter broadly, the per-
centage of HMF containing UCB tends to
increase with increasing UCB density and
decreasing temperature. This pattern sug-
gests that temperature-dependent increase
in digestion rate and passage rate of UCB
in HMF food vacuoles exceeded tempera-
ture-dependent increase in grazing rate of
HMF. For the temperature range of 13”-
26°C however, temperature-dependent de-
crease in the percentage was not evident.
From a literature value of the retention
time of particles in the food vacuoles of
HMF (20-40 min: McManus and Fuhrman
1986) and the range of average numbers of
UCB cells contained in one HMF cell (0. l-
0.4 for the UCB density of l-3 x lo5 cells
ml-l, Fig. 4), it can be roughly estimated
that an HMF cell can clear, per hour, UCB
in a volume of water which is lo5 times its
own cell volume [cf. (clearance rate of 1
HMF) = (avg number of UCB cells con-
tained in 1 HMF) x (UCB density))’ x
(retention time)- ‘1. This value suggests that
HMF in Lake Biwa graze on UCB as effi-
ciently as on bacteria (Fenchel 1982a).

510 Nagata
t
+
.-
; 60
E
40
20
0
1 ,
M J-’ J
.-,,,,--0--
- o-IOm - lo-20m --- 20970m
Fig. 3. Seasonal change of the biomass of heterotrophic microflagellates (HMF), bacteria, and unicellular
chroococcoid cyanobacteria (UCB) in the north basin of Lake Biwa. The biomass estimates are presented as
average values for the water columns of O-10, 10-20, and 20-70 m. The biomass of UCB in the deepest layer
is not indicated because it was very low (~2 pg C liter-‘).
Growth of bacteria and HMF in bottled
water-The incubation experiments were
done at temperatures ranging from 18” to
27°C. Chl a concentrations in untreated
waters ranged from 1.4 to 13.0 pg liter-‘,
of which 6.2-92.6% and 1.5-35.7% were
found in the 25-pm and the I -pm prefiltered
water, respectively. Chl a concentrations at
the beginning of the incubations were not
greatly different from those at the end of the
incubations.
Table 1 summarizes the regression pa-
rameters ofthe changes in bacterial number.
I found that bacterial density increased sig-
nificantly in the l-pm prefiltcred water ex-
cept on 24 October 1984, the density did
not change significantly in the 25-pm pre-
filtered water except on 24 August 1984 and

60 1
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Microbial food chain
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Nagata
.
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Table 2. Net production rate ofbacteria and grazing
rate of heterotrophic microflagellates on bacteria es-
timated by incubation experiments. Explanations giv-
en in text.
16 Jul 1984
24 Aug 1984
20 Sep 1984
24 Ott 1984
24 Jun 1985
8 Jul 1985
12 Aug 1985
21 Sep 1985
Production rate Grazing rate
(pg C liter I d-‘)
24 20
17
5 8
14 23
20 24
17 22
10 7
be a critical food concentration around 40
pg C liter- l below which the growth of HMF
becomes near zero.
If HMF can, in an hour, clear bacteria
from a water volume lo5 times their own
cell volume at 20°C (Fcnchel 1982a), the
clearance rate (ml HMF populations-l h-l)
can be calculated from the mean total cell
volume of HMF in the 25-pm prefiltered
water (assuming Qlo of 2). The potential
grazing rate of HMF was obtained by multiplying
the clearance rate by the mean bacterial
biomass in the 25-pm prefiltered sample.
In most cases, the independent estimates
from incubation
tent (Table 4).
experiments were consis-
The gross growth efficiency of HMF was
calculated by using the estimates of grazing
rate and net production rate of HMF
[gross growth efficiency = (net production
rate) x (grazing rate)- 1 : Table 41. The values,
depending on date and method, ranged from
11 to 53%.
Discussion
Some characteristics of the occurrence of
HMF in Lake Biwa-The densities of HMF
found in Lake Biwa ( 1 02-1 O3 cells ml-l) are
within those previously reported in some
other aquatic environments (Sieburth and
Davis 1982; Fenchel 1982~; Sherr et al.
1984; Giide 1986). Although Fenchel
(1982~) suggested that a significant fraction
of HMF is associated with particles in
Limfjorden, Denmark, I observed micro-
scopically that most HMF cells in Lake Biwa
were usually unattached. This finding and
Microbial food chain 513
3-l1c)Ic)-l@OVT
00000000
bddddddd
VVAAVVVA

514 Nagata
Table 4. Production rate (pg C liter-l d-l), grazing rate (pg C liter-’ d-l) on bacteria, and gross growth
efficiency (%) of heterotrophic microflagellates (HMF).
Grasing ratct estimated from Gross growth efficiency* estimated from
Production rate* incubation experiment biomass of HMF incubation experiment biomass of HMF
16 Jul 1984 2.1 20 7 11 30
24 Aug 1984 4.8 7 53
20 Sep 1984 8 3
24 Ott 1984 1.2 3 31
24 Jun 1985 6.0 23 12 26 50
8 Jui 1985 3.6 24 19 14 19
12 Aug 1985 8.4 22 21 38 40
21 Sep 1985 7 7
* From the slope of the regression; log (HMF density) vs. time (p) and that of the regression; log (mean cell volume of HMF) vs. time (p’) found in
Table 3. Production rate (PR) was calculated as follows: PR = CN,V[exp(24a) - I] (when $ = 0), or PR = CN,V,{exp[24@ -t r’)] - I ) (when p’
z O), where N,, and V, are initial cell density and mean cell volume, V is averaged mean cell volumes, and C is the conversion factor (71 fg C
pm ‘: Fenchcl and Finlay 1983).
t Explained in text.
$ Gross growth efficiency was estimated by dividing HMF net production rate by HMF grazing rate. For the cases of 24 August and 24 October
1984, in which UCB biomass was large (corresponding to -30% of the bacterial biomass) and the frequency of HMF containing UCB was high
(-30%), the UCB biomass was incorporated in the calculation on the assumption that HMF mgest UCB as efficiently as bacteria. Mean values of
gross growth efficiency were 23% by the incubation experiment and 37% by the biomass of HMF.
the fact that most bacteria are unattached
in this water body (see above and Nagata
1987) suggest that the prevailing mode of
trophic interaction between HMF and bac-
teria is one between unattached forms of
HMF and unattached bacteria.
An interesting vertical zonation pattern
of HMF in Lake Biwa is that HMF are more
abundant in the metalimnion than in the
epilimnion during summer stratification
(Figs. 2, 3). It is not known why this meta-
limnetic peak of HMF was found. It is dif-
ficult to explain from the vertical distribu-
tion of bacterial production rate, because I
have demonstrated that bacterial biomass
and production rate (measured by the thy-
midine method of Fuhrman and Azam
1982) decreased rapidly from the bottom of
the epilimnion to the metalimnion in Lake
Biwa (Nagata 1987). In Lake Dalnee, So-
rokin and Paveljeva (1972) reported that
the peak of the vertical distribution of HMF
moved from the epilimnion to the hypolim-
nion as thermal stratification developed.
Steinberg et al. (1983) observed in some
lakes that a species of HMF (Phyllomitus
apiculatus) was distributed more abun-
dantly in the hypolimnion than in the epi-
limnion during summer stratification. Ef-
fects of light and grazing pressure on HMF
in the epilimnion may be related to the ver-
tical zonation pattern of HMF in thermally
stratified lakes.
Evaluation ofproduction and grazing estimates
-The bacterial production rate of
5-25 pg C liter-’ d--l (Table 2) is within the
levels reported in other meso- and oligotrophic
lakes (Jordan and Likens 1980; Glide
et al. 1985; Scavia et al. 1986). In 1986, I
examined the seasonal change of planktonic
bacterial production rate in the same water
body by the thymidine method. The range
in production rate estimated with a conversion
factor of 2 X 1 O6 cells pmol- ’ was
5-60 pg C liter-l d-l in warm months (Nagata
1987). This value is close to the range
reported here.
Sherr et al. (1984) determined the growth
of HMF populations in bottled coastal seawaters
prescrecned with a 20-pm screen. The
doubling time of HMF biomass (9.7-26.5
h) and the production rate (2.4-19.0 pg C
liter-l d-l) are close to the ranges of the
present study (Table 4). In their experiments,
both bacterial density (2-6 X lo6
cells ml- I) and water temperature (19”-27°C)
were also similar to the conditions of my
experiments. As Sherr et al. pointed out,
however, larger HMF and ciliates may graze
on smaller HMF. Therefore, production
rates estimated by the present method may
be conservative.
.
The grazing rate estimated by the incubation
experiments is consistent with the
potential grazing rate of HMF estimated
from the biomass of HMF (Table 4) and

with other studies in the literature (Wright
and Coffin 1984; Glide 1986), supporting
results that bacterial production almost
equals the grazing of HMF (Table 2).
The trophic interaction between HMF and
bacteria -Although tight trophic interac-
tion between HMF and bacteria in the epi-
limnetic water was suggested by the incu-
bation experiments, no apparent seasonal
patterns with respect to HMF and bacterial
biomass coupling were found in the field
(Fig. 3). Fenchel (1982~) found that HMF
density increases following increases in bac-
terial density in Limfjorden, Denmark, sug-
gesting a tight coupling between the two.
Andersen and Fenchel (1985) have also re-
ported coupled oscillations in 8-pm pre-
screened coastal seawaters contained in
tanks. Since these prey-predator oscilla-
tions had periods of weeks, it is possible that
they could not be detected in my study
(weekly sampling). The degree of seasonal
variation in HMF biomass in the epilim-
nion (Fig. 3), however, seems to be rather
small when its potentially high growth rate
(Table 3) is taken into account. It seems
likely that HMF was consumed intensively
by plankters such as crustaceans, rotifers, or
ciliates, and HMF biomass was kept stable.
To evaluate whether food biomass was a
factor that controls the dynamics of HMF
in the field, it is useful to know the minimal
food concentration at which HMF can grow.
From experiments on several isolated strains
of HMF fed cultured bacteria, Fenchel
(1982a) suggested that this minimum bac-
terial density is 0.5-2 x lo6 cells ml-l (50-
200 pg C liter-‘). My study indicates that
the critical food concentration for natural
populations of HMF in Lake Biwa is about
40 pg C liter- ’ at water temperatures of 17”-
28°C (Fig. 5). This value is close to the lower
one of Fenchel. During warm seasons, epi-
limnetic picoplankton sometimes decrease
to this level (Fig. 3), suggesting that HMF
become starved and incapable of growth.
As suggested by Fenchel (1982b), HMF
populations may persist during such a fam-
ine by adaptive responses such as lowering
the metabolic rate or forming cysts.
Gross growth qficiency of HMF-Gross
growth efficiency of natural populations of
HMF fed on natural picoplankton was es-
Microbial food chain 515
timated in the present study (Table 4). Since
the net production rate of HMF may be
somewhat underestimated (see above), the
efficiency may also be underestimated. Al-
though estimates of efficiency varied bc-
tween methods (Table 4), and it was difficult
to determine the factors controlling efficien-
cy, the average (23 or 37%) and the range
(1 l-53%) were roughly similar to currently
reported gross growth efficiencies estimated
for cultured HMF strains fed on bacteria
(24-45%: Sherr et al. 1983; 34-43%: Fen-
chel 1982a; 39-49%: Caron et al. 1985), on
diatoms (35-62%: Caron et al. 1985; 41-
45%: Caron et al. 1986), and on bacteria
and diatoms (35-62%: Caron et al. 1985).
These results suggest that HMF in Lake Biwa
produce body carbon as efficiently as iso-
lated strains cultured under near-optimal
conditions.
Food web implications--In Lake Biwa, it
has been reported that planktonic bacteria
are not only significant components of par-
ticulate organic carbon (6-l 3% of total par-
ticulate organic carbon is attributable to
bacteria: Nagata 1986a), but also are sig-
nificant secondary producers (at least - 30%
of primary production is channeled through
bacteria: Nagata 1987). Moreover, the con-
tribution of algal picoplankton in late sum-
mer is significant (Chl a passing through a
3-pm pore-size filter corresponds to

516 Nagata
tern rather than as a link. To answer such
a “link vs. sink” argument about the mi-
cro bial loop in Lake Biwa, we must evaluate
the fate of HMF.
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Submitted: 28 May 1987
Accepted: 8 February1 988
Revised: 18 April 1988