ELSEVIER
FEMS Microbiology Ecology 20 (1996) 137- 147
Bacterivory by the ciliate Euplotes in different states of hunger
M&hail V. Zubkov, Michael A. Sleigh
Department
of Biology, lJnir;ersi&
*
ofSouthampton, Bassett Crescent East. Southampton SO16 7PX. UK
Received 20 November 1995; revised 15 February 1996: accepted 16 February 1996
Abstract
The feeding of the marine ciliate Euplotes mutubilis was studied using bacteria (Vibrio natriegens) doubly labelled with
3H-thymidine and 14C-leucine. In the presence of abundant bacteria (30 X 10” bacteria mll ’ ). an average Euplotes cell
(initially without food vacuoles) with a protein content of 12 ng consumed 16 X IO3 bacteria in the first hour and 27 X lo3
bacteria over four hours, accumulating about 60% of the bacterial protein into ciliate macromolecules.
Euplotes which had
been starved or under-fed to reduce cell protein biomass to 7 or 9 ng consumed significantly fewer bacteria, but the gross
growth efficiency for protein did not change. The rate of consumption of bacteria by large Euplotes of protein content 15 ng
was initially less than that of 12 ng cells, and it decreased markedly before the end of a 4-hour experiment. Recently divided
cells ingested bacteria rapidly, but showed a reduced gross growth efficiency of about 40%. At low bacterial concentrations
(6 x lo6 bacteria ml-‘) the rates of ingestion were markedly reduced to between + and + of maximal levels; the smallest
cells could not sustain feeding activity at the low prey concentration and gross growth efficiency fell from 43 to 20% during
a 4-hour experiment. The strategy adopted by Euplotes in response to local fluctuations in food supply involves rapid
consumption with high growth efficiency in times of plenty, but slow shrinkage without cell division to survive in times of
shortage.
Keyvordst Protozoan grazing; Bacteria consumption; Gross growth efficiency: Starvation; Microbial loop: Dual radioactive-labelled
bacteria
1. Introduction
Grazing by heterotrophic protists is believed to be
the main trophic pathway whereby the biomass produced by bacteria, cyanobacteria and minute eukary-
* Corresponding author.
otic algae enters the food web [1,2]. Microorganisms
do not live in a constant environment,
and their
concentrations
are variable, depending on the availability of suitable organic and/or
inorganic nutrients. These variations in time and space provide very
patchy food resources for phagotrophic protozoa. In
order to survive in such an unpredictable
world
protists must adapt by changing their physiological
state, to maximise the benefit they obtain from local
high concentrations
of food and to maintain themselves when prey concentrations
are low [2].
0168~6496/96/$15.00 Copyright 0 1996 Federation of European Microbiological Societies. Published by Elsevier Science B.V.
PII SO168-6496(96)00018-9
138
M.V. Zubkw,
M.A. Sleigh / FEMS Microbiology
To what extent do the ingestion and assimilation
rates of protozoa in natural environments
respond to
changes in the concentration of their food and also to
changes in their physiological state?
Ingestion rates of protists have been found to
increase hyperbolically with increase of prey concentration [3-81. Ciliates cease to divide when a certain
lower threshold concentration of prey is reached, but
they do not cease to feed [9]. The feeding rates of
protozoa are also dependent upon the size, growth
rate and physiological state of the protists [3,5]. The
efficiency
of digestion and assimilation
of prey
biomass and hence the ‘gross growth efficiency’ or
‘yield’, defined as growth (production) divided by
ingestion,
is presumed to vary not only among
species, but also according to the qualitative nature
of the food, feeding rate and temperature [6.10,1 I].
This means that both the ingestion and assimilation
of phagotrophic protists must be expected to vary
depending on their physiological state as well as the
concentration of food.
To determine and compare the dynamics of these
processes in bacterivorous
protists under different
conditions, it is necessary to study both the rate of
grazing by protists on bacteria and the rate of assimilation of prey biomass into these protists simultaneously. We have developed a method for this purpose,
based on dual radioactive labelling of bacterial prey
with 3H-thymidine
and “C-1eucine. which depends
upon the different rates of digestion and assimilation
of labelled macromolecules
of different classes [ 121.
In that preliminary
study, Euplotes ingested about
20 X lo3 bacteria in four hours; 50% of the “C label
from these bacteria was present in macromolecules
in the ciliate, but only 5% of the “H label from the
eaten bacteria was found in macromolecules
in ciliates. The rate of disappearance of ‘H could therefore
be used to estimate the rate of ingestion of bacteria,
while the rate of accumulation of “C from bacteria
gives an estimate of the assimilation of prey biomass;
as a result it is possible to perform grazing experiments without separating
protists from bacteria
[12,13].
The aim of the present study is to show how the
ingestion and growth rates of protists respond to prey
concentration in varied ways which depend upon the
physiological
history and survival strategy of the
predator. To create different physiological
states of
Ecology
20 (1996)
137-147
protists, the hypotrich ciliate Euplotes was treated
with ‘feast and famine’ stress [14] in batch cultures.
2. Materials
and methods
2. I. Culture and experimental
medium
An artificial seawater was used in culturing the
protists and in the experimental media in this study.
The composition
of this seawater was: 423 mM
NaCl, 9 mM KCl, 9.27 mM CaCl,. 22.9 mM MgC12,
25.5 mM MgSO,, 2.15 mM NaHCO,.
2.2. The culture, selection, enumeration
actice labelling of bacterial prey
and radio-
The bacterium Vibrio natriegens, originally isolated in Denmark, was grown axenically on plates of
Difco marine agar at 10°C for 2 to 5 days. Bacteria
from these plates were suspended in filtered (0.2 pm
Millipore) artificial seawater and centrifuged at 2000
rpm for 15 min at 10°C to remove clumps, but leave
suspended bacteria of a standard size range between
0.6 and 2.2 pm in length. The upper half of the
supematant was removed and provided a suspension
of these standardized bacteria for later work. The
protein content of these cells, which was measured
by the bicinchoninic
acid method (BCA) 1151, was
0.19 5 0.02 pg protein cell-’ (mean * S.D., n = 14).
Concentrations
of bacteria were monitored by absorbence at 420 and 540 nm. Accurate determinations of the concentrations
of bacteria were made in
each grazing experiment by counting on filters under
an epifluorescence
microscope. To prepare each filter. five subsamples were pooled, fixed with 2.5%
glutaraldehyde, stained with 2 mg 1~ ’ 4,6-diamidino2-phenylindole
(DAPI) and filtered onto a black 0.2
pm pore size polycarbonate
membrane.
About
1200-2000 bacteria were counted in 30 to 70 random fields at a magnification of 1250 X .
Bacteria with dual radioactive labels (DRLB) were
prepared for measurement of ingestion and accumulation rates of ciliates by Iabelling Vibrio with “Hthymidine and lJC-leucine [ 121. Briefly, suspensions
of 5 to 50 X lo6 bacteria ml -’ were prepared in the
way described above. Samples of the suspension
were incubated at 10°C with 100 nM, 1 pCi ml-’
M. V. Zubkoc, M.A. Sleigh / FEMS Microbiology
(final concentration)
[methyl-3H]thymidine
(specific
activity, 41-49 Ci mM_‘) and simultaneously
with
1 p,M, 60-70 nCi ml-’ (final concentration) leucine
L-[ “C(U)] (specific activity, 3 lo-325 mCi mM- ’ 1.
After 40-60 min the labelling was terminated by
addition of non-radioactive
analogues,
which reduced the specific activity 450-900 times. Labelled
bacteria were introduced into the containers with
ciliates about 40-60 min after dilution of the isotopes. Not only were specific molecules of the bacteria labelled, because of the short labelling period, but
similar levels of 3H and similar levels of 14C were
present in all bacteria, as demonstrated by the fact
that subsamples
of different
volumes
contained
amounts of both labels in direct proportion to the
numbers of bacteria. We call this identical labelling,
to distinguish it from uniform labelling which describes a situation when all cellular compounds contain the label in proportion to the quantity of the
element in these compounds.
2.3. The culture,
ciliate predators
enumeration
and preparation
of
Clones of the hypotrich ciliate Euplotes mutabilis
were established from single cells isolated from samples collected on the seashore of the Solent channel
near Southampton. To maintain cultures of ciliates a
100 ml aliquot of a suspension of Vibrio at about
lo8 bacteria ml-’ m
. a glass crystallizing dish was
inoculated with several cells of Euplotes and incubated in the dark at lO”C, which is approximately the
mean annual temperature of the local marine environment from which the protozoa were isolated.
Euplotes mutabilis was large enough to be picked
out individually and counted.
Ciliates for experimental
use were prepared in
different physiological
states by giving excess of
food or by starvation and subsequent feeding. To
determine the physiological state, a principal indicator of which is cell biomass, we measured the average protein content of the ciliates by the BCA
method. For each of these measurements
two replicate samples of five hundred Euplotes were picked
up individually under a dissecting microscope with a
fine-tipped Pasteur pipette.
The variety of physiological
states of Euplotes
populations were achieved by sudden exposure to an
Ecology 20 f 1996) 137-147
139
increase or decrease in food resources. While the
ciliates were concentrated at the bottom of the culture, the main part of the water was carefully aspirated off and replaced with pure seawater or bacterial
suspension. Such treatments also helped to render the
physiological state of the ciliates uniform. Five physiological states of Euplotes were identified. Four of
them were different biomass classes: about 7, 9, 12,
and 15 ng protein ciliate-‘, fed to different extent. In
order to obtain the smallest Euplotes the ciliates
were starved for a week. The fifth state was represented by recently divided Euplotes with a biomass
of about 11 ng protein ciliate-‘.
After the ciliates were counted into experimental
containers they were preincubated at 10°C for 2 h.
The purpose of preincubation
was to complete the
digestion of any food that remained in food vacuoles
and to obtain ciliates of a certain biomass without
digestive vacuoles. Protein measurements
made immediately after pipetting and over 4 h of incubation
without bacteria did not reveal any decrease in the
protein content of Euplotes that initially contained
15 ng of protein ciliate-‘.
2.4. Grazing experiments
Control samples containing only labelled bacteria
were set up in the same types of containers in an
identical manner to every experimental sample, except for the absence of ciliates. These controls were
incubated, fixed and processed in an identical manner to, and at the same time intervals as, the samples
containing
ciliates. The bacteria showed neither
growth nor any significant change in radioactivity
during the period of the experiments
[12]. In all
cases, each sample, whether experimental
or ungrazed bacteria control, was incubated in a separate
container.
Usually 400 Euplotes cells were counted into
embryo dishes in about 15 p.1 of seawater. After
preincubation
of the ciliates, bacteria that had been
labelled during the previous 2 h were added to the
experimental
dishes in concentrations
selected to
make a total volume of 1 ml. Most experiments
involved the presentation of bacterial food at about
6 X lo6 bacteria ml-’ or 30X lo6 bacteria ml-‘.
Samples (each one comprising the whole contents of
one dish) were taken at l-h intervals over a period of
140
M.V. Zubkm. M.A. Slriglz/ FEMS Microbiology Ecology 20 (1996) 137-147
4 h after addition of bacteria to follow the time
course of grazing (a few shorter experiments with
intervals between sampling of 7.5 min were used to
follow the initial accumulation of labels). The incubation was stopped by addition of 5% trichloroacetic
acid (TCA) in seawater to a final concentration
of
1%. Euplotes were removed from the 1% TCA
solution by individual pipetting into tared vials and
weighed in order to determine the volume of water
containing uneaten labelled bacteria pipetted with the
ciliates. The volume of remaining bacteria left after
removal of ciliates was also measured by weighing.
The ciliates and the remaining part of the sample
(uneaten bacteria) were separately made up to a final
concentration
of 5% TCA. After extraction for at
least 15 min at room temperature the samples were
filtered through GF/F filters (Whatman),
and radioactivity incorporated into cellular TCA-insoluble
material retained by the filter was counted with a
liquid scintillation
counter (as described in [12]).
This treatment with TCA dissolves small organic
molecules, so that the radioactive counts recorded
are those due only to the labels incorporated
in
macromolecules
[ 16,171. The concentration
of uneaten bacteria was similar in the water pipetted with
ciliates and in the remaining part of the sample; the
radioactivity stored in pipetted bacteria could therefore be subtracted from the radioactivity of the whole
subsample containing ciliates to give the amount of
radioactivity accumulated by ciliates. The accumulation of “C and 3H in the ciliates was determined. In
longer-term experiments it was possible also to measure grazing rates by monitoring
the reduction in
number of bacteria in the medium.
Grazing experiments were conducted to test the
effect of the physiological
state of ciliates on their
ingestion and growth rates. The amount of radioactive label measured in a ciliate represents the sum of
label already incorporated in macromolecules
of the
ciliate (M,) plus the label remaining
in macromolecules of ingested bacteria (M,) (the latter can
be subsequently either incorporated in ciliate macromolecules or lost by egestion or respiration). Since it
is difficult to separate these two pools of macromolecules by monitoring the radioactivity in a continuously feeding ciliate, we decided that we should
try to avoid misunderstanding
by using the term
accumulation to characterize the increase of label in
the protozoan predator (M, + M,) during the processing of its food. However, it is clear that at the
end of an experiment lasting several hours the label
remaining in bacteria within food vacuoles will have
become small compared with the label incorporated
in ciliate macromolecules
(M, -=z M,) and the overall accumulation
of label in ciliates represents production, since it is essentially
assimilation
minus
respiration. Most of the grazing experiments lasted
several hours and an equilibrium of gains by ingestion and losses by egestion and respiration (and
therefore an equilibrium of the proportion of labels
retained in the ciliate) commenced
after about an
hour.
For comparison
of the data we expressed the
amount of label accumulated in the ciliates in terms
of bacterial equivalents; i.e. a count is represented by
the number of bacteria having the equivalent total
count of that isotope in the parallel control bacterial
suspension in the similar container but without ciliates. We also introduced a term ‘accumulation efficiency’-the
amount of lJC or ‘H label (bacterial
equivalents) accumulated divided by the amount of
bacterial equivalents of that label ingested (I) per
ciliate (CM, + M,)/I).
By the end of an experiment
lasting several hours, M, < M,, and (M, + M,)/I
effectively becomes the gross growth efficiency for
particular macromolecules
(see Section 4).
2.5. ModrIling
and statistical analysis
All the experiments were designed using the principles of full factorial experiments (l3E) [l&20], in
which the effects of a number of different factors are
investigated simultaneously.
The treatment consists
of all combinations
that can be formed from the
different factors. In this case, the amount of ingested
bacteria and ‘“C or/and 3H accumulation by ciliates
(in bacterial equivalents)
were the subjective responses. The quantitative factors were: time of grazing, concentration of bacterial prey, biomass of ciliates (protein content) and concentration
of ciliates.
Digestion is a qualitative factor with two distinctive
levels; for example, in most experiments the high
level was represented by the number of ingested
bacteria and the low level by the 14C accumulation of
bacterial biomass in the ciliates, but in the shortduration experiments the high level was lJC accumulation and the low level 3H accumulation.
M. V. Zubkor, M.A. Sleigh/FEW
The results of FFE can be approximated
by a
multiple polynomial model in the form of a regression equation of the type described by Zubkov and
Sleigh [13]. The coefficients in the equation have
been calculated using Chebishev’s orthogonal polynoms and Yates’ method [19,20]. Such a complete
model precisely reproduces the original data but also
contains additional information which may or may
not be statistically significant. Two statistical methods were used to test the significance of coefficients.
The coefficients or transformed effects were compared with a normal distribution
on a half-normal
plot, and those coefficients or effects that did not fall
on a straight line passing through the origin were
regarded as statistically significant [20,21]. Also the
significance
was checked using a r-test, and only
coefficients for which t was above the critical value
were used in the regression model. The adequacy of
the model was checked using an F-test 1181. The
confidence limit chosen for all statistical tests was
95%. An average value is presented as a mean f S.D.
unless indicated otherwise, and n = number of replicates. T-tests were used to compare means.
On the graphs the vertical bars indicate f twice
the sample standard deviation (2s,- i). Replicate values are not available in all cases. Where replicates
were present the bars represent me actual value of
2s,_ , ; in other cases the bars represent the average
2s,_ , of the series of experiments
if only several
treatments of the FFE were replicated. Time zero
was not used in analysis because the value at this
time is not a variable. The solid lines on all the plots
represent the regression approximation of the experimental data; dotted lines indicate presumed trends in
the interval from the origin to the first point.
3. Results
3.1. Short-term
experiments
To gain a better understanding of the processes of
digestion and accumulation
of bacterial biomass by
protists we require information from experiments in
which the uptake of isotopes by the predator is
followed within the first hour. The consumption and
digestion of bacteria were detectable immediately
after addition of DRLB to ciliates, without any per-
A4icrobiolog.v Ecolog! 20 (1996) 137-147
0.0
7.5
15.0
Time,
141
22.5
30.0
37.5
45.0
minutes
Fig. I. Result of a representative experiment showing the accumulation of macromolecules
labelled with lJC (triangles) and ‘H
(squares), expressed in bacterial equivalents (see text), in Euplates with cell biomass of about 10 ng protein cell- ’ feeding on
bacteria at a non-limiting concentration
of about 30X 10” cells
ml-‘.
ceptible lag. In short-term experiments
where the
first sample was taken as early as 7.5 min after the
addition of bacteria to the ciliates, the amount of
accumulated j4C (1.49 f 0.46 X lo3 bacterial equivalents ciliate- ’ , n = 5) was already significantly
higher than the amount of accumulated 3H (0.337 &
0.395 X lo3 bacterial equivalents ciliate-‘.
n = 51
(Fig. 1). The accumulation of 3H and “C was linear,
with rates of 2.58 X 10’ and 10.6 X 10’ bacterial
equivalents
ciliate- ’ h ‘, respectively,
compared
with an ingestion rate of about 16 X lo3 bacteria
ciliate- ’ h-r during the first hour of grazing in
longer term experiments.
3.2. Feeding of Euplotes in different states of hunger
Preliminary experiments on Euplotes of different
biomass were conducted at different bacterial and
ciliate concentrations.
As a result of these, the contrasting bacterial concentrations
and varied physiological states of Euplotes used in the definitive
series of experiments described below were selected.
The results of these preliminary experiments were
consistent with those reported below.
When feeding on a high concentration
of about
30 X lo6 bacteria
ml-’
Euplotes
grazed more
rapidly in the first hour than subsequently irrespective of their physiological state (Fig. 2a,-e,). All the
M. V. Zubkor, M.A. Sleigh / FEMS Microbiology
142
a,
7ng
b, 9ng
c, 12ng
d, 15"g
a, pdlv
30
T
IF
01234
Time, hours
Fig. 2. Summary of series of 16 experiments showing the ingestion of bacteria (circles) and accumulation of ‘“C (triangles) by
Euplores in different physiological states feeding on bacteria at a
non-limiting concentration
of about 30X lo6 cells ml-’ (upper
half, a,-e,)
and at a limiting concentration
of about 6X lo6
bacteria ml-’
(lower half, a,,-e,,).
The Euplores had a cell
biomass in ng protein cell-’
of: 7 (a); 9 (b); 12 (cl; 15 cd):
post-division (p.div.), 1 I (e).
ciliates were eating continuously
throughout each
4-hour experiment, but the rates were different. The
biggest Euplotes significantly
reduced grazing before the end of the experiment and are considered
separately below. The ingestion and accumulation
resulting
from feeding by ciliates
of different
biomasses (7, 9 and 12 ng protein ciliate- ’ ) can be
approximated by a straight line in the interval between 1 and 4 h (Fig. ?a,--c,). The rate of 14C
accumulation is lower than the rate of ingestion and
both are also proportional
to the biomass: 7 ng
ciliates with rates of 0.88 X lo3 and 2.32 X IO3 bacteria ciliate-’
h-l, respectively; 9 ng ciliates with
rates of 1.54 X lo3 and 3.0 X lo3 bacteria ciliate-’
h-l;
12 ng ciliates with rates of 2.2 X IO3 and
3.64 X lo3 bacteria ciliate-’
h-l. While the rates
were different for ciliates of different biomass, the
14C accumulation efficiency was 60 + 7% (n = 31),
irrespective of the physiological state of the ciliates.
Only for the smallest ciliates was the efficiency
slightly decreased
to 50%, presumably
through
metabolising and respiring a larger part of the previ-
Ecology 20 f 19961 137-147
ously accumulated
material to recover the losses
caused by previous long starvation.
The biggest ciliates of 15 ng protein ciliate-’ ate
about 12 X 10” bacteria over the first hour, but then
gradually reduced grazing and almost stopped feeding by the end of the experiment (Fig. 2d,). The
rates of “C accumulation
and ingestion decreased
from 3.66 X IO3 to 0 and from 4.77 X lo3 to 0.73 X
lo3 bacteria ciliate- ’ h-‘, respectively, between 1
and 4 h. The overall efficiency of 14C accumulation
remained unchanged at about 60%.
The 3H accumulation was very low at about one
tenth of the “C accumulation,
irrespective of the
physiological
state of Euplotes. The 3H accumulation by Euplotes of 9 ng protein ciliate-’
was
0.57 AI0.22 X lo3 (n = 8); of 12 ng it was 1.13 f
0.47 X lo3 (n = 12); and of 15 ng it was 0.94 _t 0.34
X lo3 (n = 8) bacterial equivalents ciliate-’
over 4
h. The first value differs significantly from the other
two, according to the t-test. The average 3H accumulation efficiency was 5.4 f 2.2% (n = 27).
Reduction of the initial concentration
of bacteria
to about one fifth of that used above (i.e. to about
6 X lo6 cells ml- ‘) reduced the number of bacteria
consumed and the amount of labels accumulated per
ciliate to approximately
one third of the previous
level (Fig. 2a,,-e,,). The 3H accumulation gradually
increased with time. Over the first hour the accumulation of 3H by ciliates of 12 ng protein was the
highest at 0.66 X lo3 bacterial equivalents ciliate-‘,
while ciliates of 9 and 15 ng protein accumulated
0.18 X lo3 and 0.39 X lo3 bacterial
equivalents
ciliate- ’ , respectively. The rate of accumulation during 1 to 4 h for ciliates of these sizes was the same at
0.104 X lo3 bacterial equivalents ciliate-’ h-‘. The
efficiency of 3H accumulation was 6.7 f 1.7% (II =
12) and was not significantly
different from the
efficiency when grazing on high bacterial concentrations.
Starved Euplotes of 7 ng protein ciliate-’
consumed the smallest amount of bacteria at low bacterial concentrations (Fig. 2a,,). The 14C accumulation
and ingestion differed significantly;
the rates decreased from 0.8 X lo3 to -0.64 X lo3 and from
1.6 X IO3 to 0.12 X IO” bacteria ciliate-’
h-l, respectively. The decrease of rates also resulted in the
fall of the 14C accumulation efficiency from 43% to
20% between the first and fourth hours. The release
M.V. Zubkor. M.A. Sleigh/ FEMS Microbiology Ecology 20 119961 137-147
of accumulated ‘“C at the end of the experiment was
probably a result of the metabolism of 14C-labelled
macromolecules
in these strongly starved cells.
There was strong similarity of grazing of the two
bigger ciliates. The rates of 14C accumulation
and
ingestion were the same for all ciliates in these size
classes at 0.74 X lo3 and 1.74 X lo3 bacterial equivalents ciliate- ’ hh’, respectively. The average 14C
accumulation efficiency of 56 f 5.9% (n = 12) does
not differ significantly
from the accumulation
efficiency of Euplotes grazing on high concentrations of
bacteria. The rates of grazing and ‘“C accumulation
of these Euplotes feeding on low concentrations
of
bacteria were 3 - f of the rates of Euplotes of 12
ng protein ciliate-’
feeding on a high concentration
of bacteria (Fig. 2~‘).
In all the experiments described above the terms
physiological state and biomass of Euplotes could be
used interchangeably,
because the ciliates were
picked from the batch culture randomly. Therefore
the average biomass of Euplotes represented the
average physiological
state of the ciliates in the
culture. Further grazing experiments were conducted
with selected Euplotes that had divided recently or
were at the latest stage of division and completed
separation during the Z-hour preincubation.
The average protein content of these post-division
Euplotes
was about 11 ng. When fed on a high concentration
of about 30 X lo6 bacteria mll ’ ciliates ate about
8.5 X lo3 bacteria over the first hour. The consumption was low and comparable with the consumption
of Euplotes starved for a week. The rates of “C
accumulation
and ingestion were 1.7 X lo3 and 4.0
X lo3 bacteria ciliate- ’ hh’, respectively.
Fed on a
low concentration
of about 6 X lo6 bacteria ml-‘,
these post-division
Euplotes consumed about 3.5 X
lo3 bacteria ciliate- ’ over the first hour (Fig. Ze,,).
The rates of 14C accumulation and ingestion between
1 and 4 h were 0.49 X lo3 and 1.26 X IO3 bacteria
ciliate-’
hh’, respectively, which is about one third
of the rates measured when the same cells were
feeding on a high bacterial concentration.
Nevertheless, the 14C accumulation efficiency of post-division
Euplotes was similar at about 40 5 5.3% (n = 161,
irrespective of whether they were grazing on low or
high bacterial concentrations.
The 3H accumulation
efficiency was also similar at about 4.4 f 1.2% (n =
16). The 3H accumulation
was linear over 4 h of
143
feeding on either high or low bacterial concentration.
Respectively the rates were 0.21 X lo3 and 0.11 X
lo3 bacterial equivalents ciliate- ’ h- ’ .
4. Discussion
Feeding by filtering ciliates is the result of two
processes: the concentration of food particles by the
filtering apparatus and the digestion of caught food.
To maximise feeding rate both processes require
unlimited access to food, and so both processes are
influenced by the concentration of food in the environment. If the filter of a bacterivorous ciliate cannot
collect bacteria quickly enough to fill the food vacuoles as rapidly as they can be formed, then the
concentration
of bacteria is the limiting factor. By
contrast, if the filtering apparatus catches bacteria
faster than the digestion mechanism can handle them,
for example because membrane
material to form
food vacuoles may not be mobilised fast enough to
accommodate the caught food or lysosomal enzymes
to digest it [22], then the concentration of bacteria is
non-limiting.
Correct interpretation of the results of
grazing experiments
depends on a knowledge of
whether the food concentration is, or is not, limiting.
The present work is one of a very few studies
(e.g. [23]) to show the fate of macromolecules
of
bacterial prey (DNA and protein, labelled with 3Hthymidine and lJC-leucine, respectively) in a ciliate
cell. The digestion
of these classes of macromolecules was different. The 3H labelled macromolecules were mainly broken down, and Euplotes
accumulated only 5% of the consumed 3H label in
macromolecules
over the period of up to 4 h. By
contrast, a substantial proportion of the consumed
14C derived from labelled bacterial macromolecules,
mainly protein, was stored in the ciliate cell and the
14C accumulation efficiency was typically more than
50%. The difference between 3H and 14C accumulation commenced within the first 10 min in ciliates
feeding on DRLB. Studies on the cycle of changes in
food vacuoles, whereby acidification occurs almost
immediately after vacuole formation and before addition of lysosomal enzymes 124,251, agree with our
observations on the rapid disintegration
of bacteria
and shrinkage
of food vacuoles
of the ciliate
Uronema, in suggesting that it is the acidification
144
M. V. Zubkm, M.A. Sleigh / FEMS Microbiology
that makes many of the labelled molecules labile.
Subsequently
ciliates use moieties of 14C labelled
macromolecules
in synthesis of protein and other
macromolecules,
but mainly release 3H moieties or
keep them as small, TCA-soluble,
molecules. The
release of 3H may also be a result of demethylation,
because the label was placed in a methyl group of
thymidine. A small part of 3H moieties is gradually
accumulated in ciliate TCA insoluble material up to
a plateau of about lo3 bacterial equivalents ciliate- ’ .
This could indicate the extent of the requirement in
synthesis of macromolecules
of Euplotes for moieties derived from 3H-labelled
bacterial
macromolecules.
The ‘H assimilation
efficiency of the mollusc
Mulinia lateralis grazing on 3H-thymidine-labelled
bacteria was significantly lower than the 35S assimilation efficiency
of the mollusc grazing on 35Smethionine-labelled
bacteria [26]. This illustrates the
similarity of digestion of bacteria by protozoa and by
metazoa and presumably
is also due to the initial
acid treatment of ingested food. Insignificant assimilation of bacterial DNA by bacterivores is a recognized fact. When nanoflagellates
(Ochromonas
sp.1
were fed on bacteria, a six-fold increase of dissolved
DNA was observed [27]. The amount of released
DNA suggested that the majority of the consumed
bacterial DNA was egested. The protozoan retention
of 3H of consumed, thymidine-labelled
bacteria was
poor. considerably
less than the gross growth efficiency of these protozoa [28]. Because of the long
duration of incubation of growing bacteria with labelled thymidine in previous work, varying in different studies from several hours till eight days [28-301,
significant amounts of label from thymidine were
undoubtedly
incorporated into cellular components
of the bacterial cell other than DNA [28]. It is
therefore likely that protozoa grazed on these bacteria accumulated
3H from a variety of bacterial
molecules rather than exclusively from DNA. That
could explain the relatively higher assimilation efficiencies recorded by these other authors in comparison with our results. The incorporation
of the “C
label at an efficiency equivalent to the gross growth
efficiency for carbon would be expected where ciliates are fed with bacteria labelled uniformly with ‘“C
in all types of organic molecules [30].
The digestion
and assimilation
of bacterial
Ecology 20 (I 996) 137- I47
biomass in a predator cell proceed simultaneously.
The similarity of the absolute rates of uptake and
disappearance
of fluorescently
labelled
bacteria
(FL,B) from food vacuoles of ciliates and flagellates
and the report that the number of ingested FLB
reached a plateau after approximately
an hour of
grazing, strongly support the presence of an equilibrium between ingestion and egestion of food particles [ 111. The amounts of the accumulated radioactive labels in our experiments after an hour of feeding are the resultant of the processes of digestion and
metabolism (both assimilation and respiration) which
intervene between ingestion and egestion in this
equilibrium. The linear increase of ingestion and 14C
accumulation after the first hour of grazing, and the
time-independence
of “C accumulation
efficiency
during 4 h. confirm the establishment of a digestion
and metabolism equilibrium.
The “C accumulation
efficiency of Euplotes in
dishes with a large bottom surface area was 60%
when it fed on bacteria at an effectively non-limiting
concentration of about 30 X lo6 bacteria ml -I, and
56% when it fed on bacteria at a limiting concentration of about 6 X lo6 bacteria ml-‘. This difference
is not statistically significant (r-test, 95% confidence
limits). The efficiency decreased from 45 to 20%
towards the end of a 4-h experiment only when the
smallest starved ciliates were fed on a limited concentration of bacteria (Fig. 2a,,). We interpret this to
mean that their metabolic requirements could not be
compensated by the amount of food consumed; the
equilibrium of digestion and metabolism broke down
and respiration of compounds containing
“C that
had already been accumulated became necessary.
The efficiency at non-limiting food concentration
was constant and similar for ciliates ranging in
biomass from 7 to 15 ng protein celll’, except for
ciliates which were used in experiments soon after
their division, when the IJC accumulation efficiency
was significantly lower at about 40%. Probably they
needed more energy to maintain
the cell. and
metabolised a larger part of the consumed bacteria.
Significantly
lower 14C accumulation efficiencies of
22-30% were measured also when big ciliates (15
ng protein cell-‘) were grazing in a tube with low
bottom surface area on bacteria at concentrations
of
2-8 X lo6 bacteria ml-’ (data not shown). We observed that Euplotes feeding in the dishes remain
M.V. Zubkot: MA. Sleigh / FEMS Microbiology
attached to a surface for most of the time, while
those in the tube were forced to swim for most of the
time; we deduce that the actively swimming cells
require greater metabolic effort to collect their food,
and presumably need to metabolize a higher proportion of ingested proteins.
The 14C accumulation
efficiency of ciliates feeding on a non-limiting
concentration
of lo-30 X lo6
bacteria ml-’ was time-independent.
The 14C accumulation efficiency estimates the efficiency of conversion of bacterial cellular protein to protozoan
cellular protein, and can be interpreted as a somatic
growth efficiency specifically
for cellular protein,
which represents about 50% of the total biomass of
microorganisms
[31]. The somatic growth efficiency
for other compounds from the bacterial cell will tend
to be less; it is known that the gross growth efficiency of Uronema for protein is 1.4 higher than for
C in general, while Strombidium has similar efficiencies for C and protein, when feeding on bacteria [32].
Therefore, the 14C accumulation
efficiency can be
related to the gross growth efficiency, recorded for
ciliates in general at 30-50% [2,33], for scuticociliates at 42% [30], at 47% [34] or 49% for C and 70%
for cellular protein [32], and in particular for Euplotes sp. at 49% for C [30]. The r4C accumulation
efficiency of ciliates feeding on low concentrations
of l-5 X lo6 bacteria ml-i, which are common in
natural seawater, becomes dependent on the physiological state of the ciliates and on features of prey
and predator concentration,
all of which are determined by the environment.
The accumulation
efficiency of a protozoan community may therefore be a
good integral index of the carbon flux resulting from
transformation
of bacterial cellular protein in the
microbial loop.
One of the major findings of this study is that a
single ciliate species has dynamic ingestion and accumulation responses to prey abundance with respect
to the physiological
state of the predator. Just as
Choi [3] found with flagellates, smaller ciliates ingested fewer bacteria than bigger cells at the same
concentrations
of prey and predator. In the wider
range of physiological
states of Euplotes that were
studied it was revealed that the biggest ciliates used
in experiments were catching less bacteria; presumably because they were not very hungry, having been
well-fed in the recent past. Ingestion and accumula-
Ecology 20 (1996) 137-147
145
tion rates may also decrease over the duration of a
grazing experiment; this is not a result of numerous
independent
functional
responses [3], but can be
shown in experiments at non-limiting
food concentrations to be an integral response of a predator due
to its particular physiological state. For example, big
Euplotes of about 15 ng protein cell-’
ingested
bacteria and accumulated 14C actively over the first
hour, but almost ceased eating after three hours (Fig.
2dr). This can be confirmed independently
by the
number of uneaten bacteria left in the water and the
amount of 14C accumulated in the ciliates. Visually,
these Euplotes changed behaviour, and after dispersing over a dish at the beginning of the experiment,
they concentrated in the deep middle of the dish and
reduced motility towards the end of the experiment.
This was an integral functional response of a ciliate
that had consumed 20 X lo3 bacteria ciliate-’
and
accumulated 14C label equivalent to 12 X lo3 bacteria (about 2 ng of bacterial protein), which was
probably enough for it to progress to another physiological state (cell division).
Euplotes has been reported to consume efficiently
particles in the range 2.6-6.0 p,rn [9]. The Euplotes
mutabilis used in this study successfully feeds and
grows on bacteria in the 0.6-2.2 km size range. The
maximum measured clearance rate of E. mutabifis at
10°C was 1.2 p,l ciliate-’
h- ‘, which is about f of
the clearance rate of the Euplotes sp. studied by
Taylor and Sullivan [30] at 18°C. For effective filtering Euplotes need to attach to the substratum [9],
and we find that the filtration of bacteria by Euplotes
attached to the substratum appears to be three times
more efficient than that of the same ciliate in suspension. Feeding on a non-limiting
concentration
of
about 30 X lo6 bacteria ml- ’ it filtered 10 to 16 X
lo3 bacteria over the first hour of feeding, and
ingested 2-3 ng protein cell-‘, or 20-30% of the
ciliate biomass, in this time.
A threshold concentration
of bacteria does not
permit net growth of ciliates, but ciliates are still able
to feed 191. Ciliates are able to reduce bacterial
concentrations
to a low level, so that bacteria were
difficult to find in a medium containing
starved
ciliates. The results of feeding experiments showed
that even Euplotes (which is not well adapted to
planktonic feeding), was able to feed at a concentration of less than 2 X lo6 bacteria ml-‘. In a natural
146
M.V. Zubkorl. M.A. Sleigh/
FEMS Microbiology
environment
bacterial concentrations
are conspicuously variable [35]. It would be possible for ciliates,
which had successfully multiplied in a patch with a
locally high bacterial concentration,
to spread into
regions of low bacterial concentrations
and survive
there without division or growth by grazing at a low
rate, before they find another patch of rich food. This
is not the only survival strategy; another strategy,
illustrated by hypotrich ciliates, is to tolerate a long
period of starvation in the same place, by an adaptation that permits a slow shrinkage, when the prey
resource is finished. This makes sense in benthic,
especially littoral, environments,
where the sediment
is periodically
replenished
with new detritus. An
extreme result of the adaptation to a long starvation
is to encyst and form an absolutely inactive state.
However, a major cost of encystment is that when
food becomes available, the time lag before the
resumption of growth is markedly increased, so that
the organism becomes less competitive [2]. Encystment may be more adaptive for living in radically
changing abiotic environments,
for example in soils,
where all inhabitants are placed in comparable conditions.
Acknowledgements
This study was supported by the award of a Royal
Society Visiting Fellowship to M.V.Z.
References
[I] Azam, F.. Fenchel, T., Field, J.G., Gray, J.S., Meyer-Reil,
[2]
[3]
[4]
[5]
[6]
L.A. and Thingstad, F. (1983) The ecological role of watercolumn microbes in the sea. Mar. Ecol. Prog. Ser. 10,
257-263.
Fenchel, T. (19871 Ecology of Protozoa. The Biology of
Free-Living Phagotrophic Protists. Springer, Berlin.
Choi, J.W. (19941 The dynamic nature of protistan ingestion
response to prey abundance. J. Euk. Microbial. 41. 137- 146.
Curds, C.R. and Cockbum, A. (1968) Studies on the growth
and feeding of Tefruhymena pyrifomis
in axenic and
monoxenic culture. J. Gen. Microbial. 54, 343-358.
Curds, C.R. and Cockbum, A. (1971) Continuous monoxenic
culture of Tetrahymena pyriformis. J. Gen. Microbial. 66,
95- 108.
Fenchel. T. (19821 Ecology of heterotrophic microflagellates,
Ecology 20 (1996) 137-147
II. Bioenergetics and growth. Mar. Ecol. Prog. Ser. 8. 225231.
[7] Rivier, A.. Brownlee. D.C., Sheldon, R.W. and Rassoulzadegan, F. (1985) Growth of microzooplankton:
a comparative
study of bacterivorous zooflagellates
and ciliates. Mar. Micrab. Food Webs 1, 5 l-60.
[S] Taylor, W.D. (1978) Growth response of ciliate protozoa to
the abundance of their bacterial prey. Microb. Ecol. 4. 207214.
[9] Fenchel, T. (1986) Protozoan filter feeding. Prog. Protistol.
I, 65-l 13.
[lo] Fenchel, T. and Finlay, B.J. (1983) Respiration rates in
heterotrophic, free-living protozoa. Microb. Ecol. 9, 99-122.
[I 11 Sherr. B.F.. Sherr. E.B. and Rassoulzadegan.
F. (19881 Rates
of digestion of bacteria by marine phagotrophic
protozoa:
temperature
dependence.
Appl. Environ. Microbial.
54.
1091-1095.
[12] Zubkov. M.V. and Sleigh, M.A. (1995) Ingestion and assimilation by marine protists fed on bacteria labelled with radioactive thymidine and leucine estimated without separating
predator and prey. Microb. Ecol. 30, 157- 170.
by
[I31Zubkov, M.V. and Sleigh, M.A. (19951 Bacterivory
starved marine heterotrophic nanoflagellates
of two species
which feed differently, estimated by uptake of dual radioactive-labelled bacteria. FEMS Microbial. Ecol. 17, 57-66.
[141Koch, A.L. (1971) The adaptive responses of Escherichin
coli to a feast and famine existence. Adv. Microb. Physiol. 6,
147-217.
1151Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K.,
Gartner. F.H., Provenzano, M.D.. Fujimoto. E.K., Goeke.
N.M.. Olson, B.J. and Klenk, D.C. (1985) Measurement of
protein using bicinchoninic acid. Anal. Biochem. 150, 76-85.
ll61 Fuhrman. J.A. and Azam. F. (1982) Thymidine incorporation
as a measure of heterotrophic bacterioplankton
production in
marine surface waters: evaluation and field results. Mar.
Biol. 66, 109-120.
J.P. and Bouvy, M. (19911 Estimating bacterial
1171To&ton,
DNA synthesis from [ ‘Hlthymidine incorporation: discrepancies among macromolecular
extraction procedures. Limnol.
Oceanogr. 36. 299-306.
[I81 Adler, Y.P.. Markova, E.V. and Granovsky, Y.V. ( 1976)
Design of Experiment to Optimise Conditions, 2nd ed. Nauka
tin Russian).
[I91 Cochran, W.G. and Cox, G.M. (1957) Experimental Designs,
2nd ed. Wiley, New York.
DO1Maximov, V.N. (1980) Multifactorial Experiment in Biology.
Moscow State University (in Russian).
I211 Draper, N.R. and Smith, H. (19811 Applied Regression Analysis, 2nd ed. Wiley, New York.
WI Nilsson. J.R. (1976) Physiological and structural studies on
Tetrahymma priformis
GL. C. R. Trav. Lab. Carlsberg 40.
215-355.
Techniques: LocalizaD31 Gude, W.D. (1968) Autoradiographic
tion of Radioisotopes
in Biological Material. Prentice-Hall,
Englewood Cliffs, N.J.
in furumecium.
Biol.
[241Mast. S.O. (1947) The food-vacuole
Bull. 92. 31-72.
M. V. Zubkor, M.A. Sleigh / FEMS Microbiology Ecology 20 (1996) 137-147
1251 Fok, A.K. and Allen. R.D. (1988) The lysosome system. In:
Paramecium
(GGrtz, H.-D., Ed.), pp. 301-324.
Springer,
Berlin.
[26] Chalermwat, K.. Jacobsen, T.R. and Lutz, R.A. (1991) Assimilation of bacteria by the dwarf surf clam Mulinia lateralis. Mar. Ecol. Progr. Ser. 71. 27-35.
[27] Turk, V., Rehnstam, A-S., Lundberg, E. and Hagstrom, A.
(19921 Release of bacterial DNA by marine nanoflagellates,
an intermediate step in phosphorus regeneration. Appl. Environ. Microbial. 58, 3744-3750.
[28] Caron, D.A., Lessard, E.J., Voytek, M. and Dennett, M.R.
(1993) Use of tritiated thymidine (Tdrl to estimate rates of
bacterivory:
implication of label retention and release by
bacterivores. Mar. Microb. Food Webs 7, 177-196.
[29] Hollibaugh, J.T.. Fuhrman, J.A. and Azam, F. (19801 Radioactively labelling of natural assemblages of bacterioplankton for use in trophic studies. Limnol. Oceanogr. 25, 172181.
[30] Taylor, G.T. and Sullivan. C.W. (1984) The use of ‘“C-
[31]
[32]
[33]
[34]
[35]
147
labelled bacteria as a tracer of ingestion and metabolism of
bacterial biomass by microbial grazers, J. Microbial. Methods 3, 101-124.
Ingraham,
J.L., Maaloe, 0. and Neidhardt,
F.C. (19831
Growth of the Bacterial Cell. Sinauer Associates.
Ohman. M.D. and Snyder, R.A. (1991) Growth kinetics of
the omnivorous oligotrich ciliate Sfrombidium sp. Limnol.
Oceanogr. 36, 922-935.
Bernard, C. and Rassoulzadegan,
F. (1990) Bacteria or microflagellates
as a major food source for marine ciliates:
Possible implications for the microzooplankton.
Mar. Ecol.
Prog. Ser. 64, 147-155.
Sherr. B.F., Sherr, E.B. and Fallon, R.D. (19871 Use of
monodispersed,
fluorescently labelled bacteria to estimate in
situ protozoan bacterivory.
Appl. Environ. Microbial. 53.
958-965.
Mitchell, J.G. and Fuhrman, J.A. (1989) Centimeter scale
vertical heterogeneity
in bacteria and chlorophyll a. Mar.
Ecol. Prog. Ser. 54, 141-148.