Journal of Plankton Research Vol.21 no.9 pp.1791–1798, 1999
SHORT COMMUNICATION
Influence of light intensity on the ingestion rate of a marine ciliate,
Lohmanniella sp.
Kung-Ming Chen and Jeng Chang1
Institute of Marine Biology, National Taiwan Ocean University, Keelung 202-24,
Taiwan, Republic of China
1To
whom correspondence should be addressed
Abstract. Using fluorescently labeled algae (FLA) as food particles, the ingestion rate of an oligotrichous ciliate, Lohmanniella sp., was 0.4 FLA ciliate–1 min–1 in the dark, but decreased to 0.07 FLA
ciliate–1 min–1 when illuminated at 115 µE s–1 m–2. When the light was abruptly switched on or off,
changes in ingestion rate were immediate with no delay. These results suggest that the effect of light
may have to be considered in the experimental design when measuring ciliate feeding rates in marine
environments.
A large part of the phytoplankton standing stock and productivity in the sea is
due to ultraphytoplankton, species with a size smaller than 5 µm (Li and Wood,
1988). These cells are too small to be captured by mesozooplankton, but are the
major food source for microzooplankton including ciliates and flagellates (Sherr
et al., 1987; Turner et al., 1988). These protozoans regulate the community structure of ultraphytoplankton and, at the same time, channel organic material from
the microbial loop to higher trophic levels (Sherr and Sherr, 1988). Compared to
that of flagellates, the ingestion rate of ciliates on ultraplankton was higher (Sherr
et al., 1991). Thus, an accurate estimation of ciliate ingestion rates is essential to
assess how primary production of ultraphytoplankton is dispersed through the
marine food web.
The ingestion rate of individual protozoan cells is affected by many factors,
with prey size and concentration being the two most frequently mentioned (Sherr
et al., 1991). Other factors that have been shown to influence food uptake rates
include temperature, predator size and food quality (Bird and Kalff, 1987; Sherr
et al., 1987; Peters, 1994). Another potentially important environmental factor is
light. However, depending on the organisms tested or the ecosystems examined,
the relationship between protozoan feeding activities and light is so diverse that
no common trend exists. In a Paramecium containing symbiotic Chlorella, the
uptake rate of fluorescent beads was higher under higher light intensities. The
change in ingestion rate required an acclimation period, and the ingestion rate
was correlated to the concentration of endosymbiotic algae (Berk et al., 1991). In
the Bothnian Sea, bacterial grazing loss reached a daily minimum in the morning
for two consecutive days, and the rhythm was apparently caused by cessation of
feeding during the flagellate cell division period (Wikner et al., 1990). In contrast,
when the ingestion rate of a chrysophyte flagellate was measured in five lakes,
elevated consumption on bacteria occurred at night, but only in one lake (Bird
© Oxford University Press 1999
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K.-M.Chen and J.Chang
and Kalff, 1987). Diel variation in ingestion rates was absent from the other four
lakes.
Oligotrichous ciliates are major consumers of ultraplankton-sized algae in
marine environments (Sherr et al., 1991; Stramski et al., 1992). However, the
effects of light on their feeding rates have received little attention. In this report,
we investigated light-induced changes in the ingestion rate of a cultured marine
oligotrichous ciliate, Lohmanniella sp., using the fluorescently labeled algae
(FLA) technique. In addition, an experiment was designed to distinguish whether
the altered ingestion rate was caused by light or was a result of a diel feeding
rhythm.
A water sample was collected in November 1997 from a coastal station
(25°08.59N, 121°47.79E) on the northern coast of Taiwan. After returning to the
laboratory, 500 ml of the sample were screened through a 200 µm mesh to remove
macrozooplankton and then diluted with an equal amount of an algal culture. The
algal culture contained a unicellular coccoid alga, which was isolated from the
same location in January 1997, and was maintained in f/2 medium (Guillard and
Ryther, 1962) at 20°C. Every 3 days, 20% of the water sample was replaced by
the algal culture. After 5–6 days, a planktonic oligotrichous ciliate, cf. Lohmanniella sp., became the dominant protozoa in the flask (Figure 1). The ciliate has
a diameter of 20–35 µm and is morphologically similar to the Lohmanniella
described in Maeda (1997). The culture of Lohmanniella sp. was maintained
under a 12:12 h light–dark cycle at 20°C, and the 20% dilution with algal culture
was performed every week. The concentration of Lohmanniella was enumerated
daily to estimate the growth rate.
The FLA were prepared with the coccoid alga according to the procedure of
Rublee and Gallegos (1989), and used as feeding particles. The coccoid alga is
Fig. 1. General morphology of Lohmanniella sp. The cell is globular with a diameter of 33 µm. The
buccal cavity is closed with a membrane-like seal.
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Effect of light on ingestion rate of Lohmanniella sp.
eukaryotic, non-motile and has a diameter of 2 µm. The algal culture was allowed
to grow to a concentration of 106–107 cells ml–1, and cells were harvested via
centrifugation. Concentrated cells were resuspended in 10 ml of phosphatebuffered saline (PBS; 0.05 M Na2HPO4 in 0.85% NaCl, adjusted to pH 9), and
incubated for 2 h at 54°C with 2 mg of the fluorescent dye 5-(4,6-dichlorotriazin2-yl) aminofluorescein (DTAF) added. Prepared FLA solutions were divided into
1 ml aliquots and stored at –20°C as the stock solution. Shortly before a feeding
experiment, a stock solution of FLA was thawed and diluted 1:10 with PBS to
make a working stock. Immediately before use, the working stock was vortexed
for 2 min, followed by passage through a 20 µm mesh to reduce clumping.
Actively growing Lohmanniella culture with a concentration of 20–25 ciliates
ml–1 was used in all feeding experiments. Conical polypropylene centrifugation
tubes (Falcon, Franklin Lakes, NJ) were used as incubation vessels, and each
contained 30 ml of Lohmanniella culture. The feeding experiment was performed
on a laboratory bench with room temperature maintained at 25°C; the temperature was monitored by placing a thermometer in a flask filled with distilled water.
During incubation, the Lohmanniella culture was illuminated laterally by four
daylight fluorescent bulbs, and the light intensity inside the centrifugation tubes
was measured with a light meter equipped with a 4 p quantum sensor (Qsp170BD, Biospherical Instruments, San Diego, CA). With this set-up, a maximum
light intensity of 115 µE s–1 m–2 was achieved. Various light intensities were
achieved by adjustments using neutral-density filter sheets (Lee Filters, Hampshire, UK). A carefully sealed cardboard box placed on the same bench served
as the chamber for dark treatment. Before the onset of feeding experiments, a
series of centrifugation tubes containing Lohmanniella culture was left undisturbed in various light intensities for 30 min to allow the protozoa to recover from
handling shock (Sherr et al., 1987). Next, the concentration of the coccoid alga
co-existing with Lohmanniella was enumerated and the proper amount of FLA
was gently injected into the centrifugation tubes. The concentration of FLA was
adjusted to 40–50% of that of coccoid algae in the background. During the incubation period, one tube was removed every 10 min, and the sample was preserved
with sequential addition of alkaline Lugol’s solution (final concentration 0.5%),
borate-buffered formalin (final concentration 3%) and a drop of 3% sodium thiosulfate (Sherr et al., 1987). The preserved samples were stored in the dark at 4°C
until microscopic examination. All samples were analyzed within 1 week.
To enumerate FLA ingested by individual ciliates, preserved samples were
concentrated by centrifugation at 2000 r.p.m. for 10 min and the volume was
reduced to 1 ml. The concentrated sample was then placed in a Sedgwick–Rafter
counting cell and examined with a Nikon epifluorescence microscope. The Sedgwick–Rafter counting cell was first scanned under transmitted light at 3100 to
locate Lohmanniella cells. Once found, magnification was increased to 3400, and
the ciliate was examined under epifluorescent illumination to count FLA within
the cell. In each sample, 30–40 individuals of Lohmanniella were counted and the
average FLA content was used for ingestion rate calculation. The method of
linear regression was used to estimate the ingestion rate of FLA based on the
increase in mean FLA per ciliate with time (Sherr et al., 1987).
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K.-M.Chen and J.Chang
Lohmanniella sp. in culture grew at a rate of 0.32 day–1, indicating that the
coccoid alga is a proper food source for active growth. During feeding experiments, the FLA in individual ciliates varied over a wide range. For example, after
being incubated in the dark for 10 min, Lohmanniella cells were found to contain
from 0 to 16 FLA particles. However, by averaging the FLA content of 30 or more
ciliates, a precise estimate of the mean could be achieved. The standard error was
usually within 12% of the mean. During the 40 min incubation period, the mean
FLA content per ciliate increased linearly with time. We found no significant
differences in concentrations of background coccoid alga before and after the
incubation period. The regression lines usually matched the data points well with
slopes significantly different from zero (Figure 2).
The rate of increase of FLA content in Lohmanniella cells was significantly
higher in the dark (Figure 2). When the feeding experiment was performed at
noon (6 h after onset of the light period), the ingestion rate in the dark exceeded
that in the illuminated (115 µE s–1 m–2) condition by a factor of 4.5 (Table I). An
identical experiment performed at midnight (6 h after onset of the dark period)
showed the same trend (Figure 2, Table I).
To examine the response time required for Lohmanniella sp. to alter its feeding
rate in different light treatments, ciliates were first incubated in the dark for 40
min and then switched to the illuminated condition (Figure 3). In this experiment,
Lohmanniella maintained an ingestion rate of 0.33 FLA ciliate–1 min–1 in the dark
(Table II). Immediately after the lights were switched on, the FLA content in
Fig. 2. FLA ingestion rate of Lohmanniella sp. measured in dark (d) and illuminated (s) conditions.
(a) The noon experiment started 6 h after the onset of the light period. (b) The midnight experiment
started 6 h after the onset of the dark period. Error bars represent ±1 SE.
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Effect of light on ingestion rate of Lohmanniella sp.
Table I. Effect of light on the ingestion rate of Lohmanniella sp. FLA ingestion rates under
illuminated and dark conditions were measured both at noon and at midnight. Standard errors are
included in parentheses
Time of day
Ingestion rate (FLA ciliate–1 min–1)
——————————————————————————————
Illuminated
Dark
11:30
23:30
0.08 (0.02)
0.06 (0.01)
0.37 (0.03)
0.44 (0.03)
Table II. Effect of abrupt changes in light intensity on FLA ingestion rates in Lohmanniella sp.
Standard errors are included in parentheses
Date
(1997)
Treatment
Ingestion rate (FLA ciliate–1 min–1)
——–————————————————————
Before switching
After switching
4 December
Light to dark
Dark to light
Light to dark
Dark to light
0.13 (0.02)
0.33 (0.01)
0.09 (0.06)
0.32 (0.02)
10 December
0.24 (0.03)
–0.17 (0.02)
0.21 (0.04)
–0.26 (0.03)
Fig. 3. Effect of sudden changes in light intensity on FLA ingestion rates in Lohmanniella sp. The
light-to-dark switching experiment is marked by open circles (s) and the dark-to-light switching
experiment is marked by filled circles (d). Arrows mark the time when light switching occurred. Error
bars represent ±1 SE. Regression lines are fitted to various segments of the experiments and are used
to obtain ingestion rates. The upper and lower panels represent replicate experiments on 4 and 10
December 1997.
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K.-M.Chen and J.Chang
individual ciliates began to decrease with time. Conversely, if a culture was first
incubated under illuminated conditions, a low ingestion rate was observed. When
the incubation condition was switched to darkness, the ingestion rate suddenly
increased with no delay (Figure 3, Table II). A replicate experiment revealed a
similar trend.
A series of feeding experiments at various light levels was designed to determine the minimal light intensity required to inhibit feeding (Figure 4). In this
batch of experiments, the distribution of Lohmanniella was rather uneven in the
culture, which made it difficult to collect enough ciliates for FLA counting in
some centrifugation tubes. Although the uneven distribution produced larger
uncertainties in determining ingestion rates, a general trend was still observed
(Figure 4). Results of the feeding experiments (Figure 4) showed that Lohmanniella sp. had higher ingestion rates in the two sets incubated at the lowest light
intensities. The ingestion rate was close to 0.6 FLA ciliate–1 min–1 when the light
intensity was below 17 µE s–1 m–2. In contrast, when the light intensity was raised
to >57 µE s–1 m–2, the ingestion rate decreased to ~0.3 FLA ciliate–1 min–1.
The observed variations in Lohmanniella ingestion rates are apparently a
Fig. 4. FLA ingestion rate of Lohmanniella sp. as a function of light intensity. Upper two panels: mean
FLA content of individual ciliates at various time points during incubation. Straight lines were
obtained by linear regression and the number at the end of each regression line indicates the light
intensity used for incubation. Error bars represent ±1 SE. Lower panel: variation of ingestion rate at
six different light intensities.
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Effect of light on ingestion rate of Lohmanniella sp.
behavioral response to light intensity. Although it is possible that the background
coccoid algae proliferated more actively under the illuminated condition, which
in turn diluted the concentration of FLA, and caused a lower ingestion rate,
several lines of evidence do not support such a deduction. The incubation period
lasted for 40 min only. As indicated by cell counts before and after incubation,
this period is apparently too short for a significant increase in algal concentration
to occur. In addition, the ingestion rate changed immediately and abruptly in the
light-switching experiments (Figure 3). It is unlikely that the proliferation rate of
the coccoid algae could change at the same pace.
The mechanism that converts the signal of light intensity to a decreased ingestion rate in Lohmanniella is unknown. In the literature, all protozoans that have
been reported to have a light-dependent ingestion rate are either mixotrophic
flagellates or species with symbiotic algae. For example, the increased ingestion
rate of a Paramecium under light requires the presence of symbiotic Chlorella cells,
otherwise, the response to light disappears (Berk et al., 1991). In a mixotrophic
phytoflagellate, Poterioochromonas malhamensis, the ingestion rate decreases
when light intensity exceeds 400 µE s–1 m–2 (Porter, 1988). In contrast, the
inhibitory light intensity in Lohmanniella is much lower at 57 µE s–1 m–2 (Figure
4). Although we did observe red-fluorescing particles in Lohmanniella cells under
the fluorescence microscope, this is not direct evidence for mixotrophy. Another
way for protozoans to detect light is to develop an eyespot. This organelle has been
linked to phototaxis in oligotrichous ciliates (Jonsson, 1994), but Lohmanniella
cells contain no heavily pigmented area resembling an eyespot (Figure 1).
Our results imply that the ingestion rate of certain species of protozoans in
marine environments is similarly influenced by light. At our sampling site, the
Secchi depth was 3.6 m and the light intensity at noon on a sunny spring day was
1100 µE s–1 m–2. Under these circumstances, the ingestion rate of Lohmanniella
would be seriously suppressed in depths shallower than 6 m, which is 63% of the
euphotic zone thickness. If this phenomenon is common in phagotrophic protists,
we must re-evaluate the suitability of using dark incubation for measuring ingestion rates. In addition, how the current view of carbon budgets in the microbial
loop will change when light is included as a factor affecting ingestion rates at
various trophic levels deserves further investigation.
Acknowledgements
We are grateful to Dr K.P.Chiang for his help in identifying the ciliate species,
and to Yuan-Pin Chang, Su-Fen Wei and Chia-Hsing Chen for their assistance in
sampling. We also thank Drs A.Taniguchi, D.Stramski and E.B.Sherr for helpful
suggestions. This work was supported by a National Science Council (ROC) grant
NSC 87-2611-M-019-013-K2.
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Received on January 12, 1999; accepted on April 28, 1999
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