BULLETIN OF MARINE SCIENCE, 43(3): 764-771, 1988
CHEMOSENSORY RESPONSES OF HETEROTROPHIC
AND MIXOTROPHIC FLAGELLATES TO
POTENTIAL FOOD SOURCES
Susan J. Bennett, Robert W Sanders and Karen G. Porter
ABSTRACT
Chemosensory responses by freshwater heterotrophic and mixotrophic micro flagellates to
amino acids, bacteria and bacterial exudates were measured using the Van Houten T-maze
assay. Positive responses by Monas sp. I were demonstrated to 0.1 M CasAmino acids within
30 min. Bacteria resuspended in sterile water elicited no response from Monas sp. I after 30
min, but a positive response was observed after 90 min. Monas sp. 2 also showed a positive
chemosensory response to the presence of bacteria after 90 min. Strongest positive responses
by Monas sp. 2 were to bacterial exudates with or without cells present. These data suggest
that heterotrophic flagellates may be attracted to areas of high bacterial densities by chemical
cues present in bacterial exudates. The Van Houten T-maze tests also indicated a strong
positive response to a toxic bacterium, Chromobacterium violaceum. This may be due to the
death and deposition of cells upon entry in the T-maze arm with the toxins. The pigmented
mixotroph, Poterioochromonas showed no response to bacteria with or without exudates after
90 min, and a negative response after 24 h. Phytoflagellates may avoid bacteria as potential
competitors for nutrients. Alternatively, the arm containing bacteria could have become
chemically inhospitable after 24 h and the flagellates were responding to an adverse chemical
stimulus.
Heterotrophic and autotrophic micro flagellates are ubiquitous members ofmarine and freshwater plankton (Pomeroy, 1974; Fenchel, 1987; Glide, 1986). Their
abundances (- 106/1)are orders of magnitude higher than that of other eucaryotic
members of the planktonic community. Bacterivorous flagellates are important
in controlling picoplankton numbers (Fenchel, 1982; Azam et aI., 1983; Wright
and Coffin, 1984; Glide, 1986). In a seasonal study of flagellates in Lake Oglethorpe, Georgia, heterotrophic microflagellates were the dominant grazers, constituting 55-99% of bacterivory throughout the year (Sanders, Porter, Bennett,
and DeBiase, submitted) Mixotrophic phytoflagellates, (i.e., simultaneously phagotrophic and photosynthetic flagellates), were important grazers in the winter when
they were most abundant and cropped 20% of the bacterial standing stock per
day (Sanders and Porter, 1988). The greatest abundances of flagellates were associated with the metalimnetic plate, which is also a region of high bacterial
numbers and production (Bennett, Sanders and Porter, in prep.; McDonough et
aI., 1986).
In addition to macro scale differences of abundance and grazing, there are also
microscale variations in activity. Bacteria, flagellates and other protozoa are often
found concentrated in particulate matter and bacterial aggregates (Caron et aI.,
1982; Biddanda, 1985). Protozoa have the ability to orient toward nutrients, prey
items and other attractants (Antipa, 1983). This chemosensory behavior coupled
with motility would enhance the probability of encounters with microzones or
larger patches. Goldman (1984) suggested that a main function of motility in
flagellates may be to seek out enriched microaggregates. The high abundance of
flagellates in oligotrophic waters may be due to their ability to track and utilize
these micropatches.
Many assays have been developed to collect qualitative and quantitative data
defining these chemosensory behaviors in protozoa. One of the first investigations
764
BENNETT ET AL.: CHEMOSENSORY RESPONSES OF FLAGELLATED PROTISTS
765
of flagellate responses was by Fox (1921), who showed aggregation ofthe kinetoplastid Bodo in meat extract and within an optimal oxygen concentration. Hauser
et ai. (1975) observed a differential embedding response by a heterotrophic marine
dinoflagellate to various amino acids, quaternary amines and carbohydrates in
test agar solutions in Petri dishes. Spero (1985), using a microcapillary technique,
also demonstrated a chemosensory response by a phagotrophic dinoflagellate to
a variety of amino acids and other organic compounds. Using a modification of
the capillary pipet technique, Verity (1988) quantified ciliate chemosensory response as it was affected by their feeding history and the physiological state of
their prey. The T-maze assay, developed by Van Houten, quantified attraction to
potassium acetate and repulsion to quinine-Hel by Paramecium (Van Houten et
aI., 1975). Using the same assay, Sibbald et ai. (1987), reported a chemosensory
response by the marine heterotrophic microflagellate Pseudobodo tremulans to
bacteria and several nitrogen compounds.
Despite the differences in methodologies, these studies reveal similarities in
behavioral response to stimuli among ciliated and flagellated protozoans. Often
compounds eliciting the response were associated with the food source of the
organisms (Antipa et aI., 1983; Levandowsky et aI., 1984; Sibbald et aI., 1987).
It is not clear whether this is a chemotactic or a chemokinetic response. Independent of the mechanism, a chemosensory response would be ecologically advantageous to protozoans for tracking concentrations of prey items in a generally
dilute aquatic environment. In this study we utilized the T-maze to investigate
the ability offreshwater heterotrophic and mixotrophic flagellatesto track bacterial
food sources.
MATERIALS AND METHODS
Monas spp. were isolated from Lake Oglethorpe using a silicone-oil plating technique (Soldo and
Brickson, 1980). The bacterium Klebsiella sp. was added as a food source to lakewater pre-filtered
through a 20-,um sieve. The enriched, filtered lakewater (2 ml) was vortexed with 4 ml of silicone oil
and the mixture was poured into 60-mm Petri dishes. Discrete microdroplets of water containing
single flagellates were trapped in the hydrophobic surfaces of the oil. After a 24-h incubation period,
droplets with flagellate clones were transferred to culture tubes of 0.1 % (w:v) Cerophyll. The clonal
cultures were examined using epifluorescent microscopy at a magnification of 1,250 x to determine
cell size and to insure that they lacked photosynthetic pigments. Monas sp. I was 5·4,um and Monas
sp. 2 was 4·3 ,urn. Both were ovoid, had two unequal length flagella and had no auto fluorescing
pigments. The phagotrophic phytoflagellate Poterioochromonas malhamensis was obtained from the
Starr culture collection (Starr, 1978). This chrysophyte was approximately 5·6 ,urn with two unequal
length flagella. Poterioochromonas was maintained under fluorescent lights (400 !LE' m -2. S-I) in Ochromonas media (Starr, 1978).
The T-maze experiments were modelled after those of Van Houten et al. (1975). One arm of a
three-way stopcock and the space inside the stopcock were filled with the control solution. and a
second arm was filled with the test solution. Flagellates were added to the third "entry" arm, the maze
was positioned horizontally, and the stopcock was opened. After 30 min, the stopcock was closed,
the arms were emptied into separate vials, and the contents were fixed with 1% Lugols solution.
Experiments were performed in the dark to eliminate any phototaxic responses and the particular arm
of the T-maze chosen for control, test, or entry was varied randomly among replicates. Flagellates
were enumerated at 200 x in a Sedgcwick-Raftcr cell. At least 200 cells were counted per replicate. A
chemosensory index (lJ was used to characterize behavior in the T-maze. It is defined as follows: Ie
= T/(T + C), where T equals the number of cells in the test ann, and C equals the number of cells
in the control arm. Ieof 0.5 indicates no response, Ie > 0.5 indicates a positive response and Ie < 0.5
indicatcs a negative response (Van Houten et aI., 1975).
The first series of experiments investigated chemosensory responses by 1'vfonas sp. I to an amino
acid mixture. A 0.1 M solution of CasAmino acid (Difco Laboratories) was added to the test ann.
The control arm contained filtered, autoclaved lakewater. A second experiment was designed to
determine if }vfonas sp. was attracted to bacterial cells. Klebsiella sp. was grown on Cerophyll medium,
centrifuged and resuspended in 0.22-,um filtered, autoclaved lakewater, and added to the test arm.
The control arm was filled with filtered, autoclaved lakewater.
766
BULLETIN OF MARINE SCIENCE, VOL. 43, NO.3, 1988
1.00
0.25
0.00
+---------,r---------,---------,
90
60
30
120
Time (minutes)
Figure 1. Time course for Monas sp. I when tested with bacteria resuspended in sterile water. The
chemosensory index increases up to 90 min and then levels off.
After these preliminary experiments, the testing procedure was modified by: (I) changing the types
of test media, (2) testing mixotrophic flagellates, and (3) increasing the time course for response. The
test for response to bacteria stimulus was refined to determine whether bacterial cells, exudates, or
both were critical in eliciting chemosensory behavior. Aerobacter aerogenes, grown for two days on
0.1 % Cerophyll was used for these experiments. Aerobacter aerogenes and Klebsiella sp. are both 12-,um long rod-shaped bacteria and supported growth of flagellates in culture equally well. One portion
of the A. aerogenes culture was fractionated with a 0.22-,um Nucleopore filter to obtain bacteria-free
exudates, and another portion was centrifuged twice at 1,500 g for 20 min and resuspended in sterile
0.22-,um filtered tap water to minimize exudates and supply only bacteria cells. The initial culture
containing cells and exudates was also tested. Comparative assays were run using bacterial cultures
of the same age and abundances. Flagellate response to a toxic bacterium, Chromobacterium violaceum,
was also tested. C. violaceum was grown on tryptic soy agar slants, resuspended in sterile 0.1 %
Cerophyll, and allowed to grow for 12 h. The broth cultures were then filtered and centrifuged in the
same manner as the Aerobacter to isolate particles and exudates.
A second aspect of this investigation was to compare responses of phagotrophic phytoflagcllates
and zooflagellates. lo..fonassp. 2 grown on 0.1% Cerophyll with Aerobacter was centrifuged at 200 g
for 10 min to separate the flagellates from their bacterial prey and culture media. The phagotrophic
phytoflagcllate Poterioochromonas malhamensis was separated from culture media in a similar manner.
Both species were washed and resuspended in a non-organic "starvation" media (5 media; Myers and
Graham, 1956) and maintained in the dark for 24 h prior to the experiments. Microscopic observation
indicated that cells were motile and apparently healthy after this treatment.
To eliminate possible experimental artifacts, all aspects of the preparation of control and test
solutions, and bacteria and flagellate cultures were kept consistent. The sterile lake and tap water used
as control solutions or to resuspend bacteria were all from the same batches. A preliminary assay
demonstrated that 0.1 % Cerophyll did not stimulate a chemosensory response in these flagellates
relative to a control solution of sterile aged tap water. The tap water was used for a control in all
remaining experiments which allowed for a consistent comparative assay to be run using various types
of bacterial stim uli. The flagellates tested were also manipulated in batches and the same cell suspension
was used among replicates and treatments to allow for direct comparison between the various stimuli.
The bacterial cell suspensions used were from 48-h cultures and were of similar concentrations (-10'
cells ml- 1).
RESULTS
AND DISCUSSION
The initial experiment to examine chemosensory behavior in Monas sp. I
showed a significant directed response toward CasAmino acids after 30 min (Ie =
BENNETT
ET AL.: CHEMOSENSORY
RESPONSES
OF FLAGELLATED
767
PROTISTS
Table 1. Mean chemosensory indexes (± 1 SO) for Monas sp. 2 tested with the bacteria Aerobacter
aerogenes tested using the Mann-Whitney U-test (there were three replicates for each experiment)
Index of chemosensory
Exudates
Bacteria in
Exudates
Bacteria in
Bacteria in
Bacteria in
sterile water
culture media
culture media
sterile water
Significance
response
0.92
0.80
0.92
0.87
0.87
0.80
(±0.023)
(±0.027)
(±0.023)
(±0.023)
(±0.023)
(±0.027)
P < 0.025
P < 0.04
P < 0.025
0.83 ± 0.02). j!Jonas sp. 1 showed no significant chemosensory response toward
Klebsiella without exudates after 30 min. The chemosensory response was 0.59
± 0.08, which is not significantly different from the null response of 0.5. A time
course was then run and Alonas sp. 1 showed an increasingly greater response to
Klebsiella for up to 90 min (Fig. 1). There are two non-exclusive explanations for
this phenomenon. First, flagellates may require a longer time course to detect the
bacterial cells than the amino acids, due to differences in signal strength and
diffusion rates. Secondly, after 30 min there may not have been sufficient time
for the bacteria to build up excretion products to cue the flagellates. This indicates
that these flagellates have the ability to detect chemical cues in their environment
and alter their swimming behavior to accumulate in areas of higher stimuli concentration. In the experiments with freshly suspended bacteria, the 90 min time
period may have been necessary for enough bacteria exudate to accumulate in
the test arm to cue the flagellates.
With the second set of assays, the objective was to investigate the component
of the bacterial stimuli that produced the greatest chemosensory response. The
heterotrophic flagellate, Monas sp. 2, demonstrated a positive chemosensory response to all tests with bacterial cells and/or exudates after 90 min. When presented
the food bacterium Aerobacter, they showed a greater response to intact cultures
or filtrates than to bacteria in autoclaved tap water (Table 1). This suggests that
exudates rather than the particles themselves are most important in eliciting a
response. In the same type of assay, Antipa et a1. (l983) demonstrated that Paramecium and its predator Didinium were both attracted to bacteria in growth
media, to a filtrate of the culture, and to a chemoattractant isolated from the
bacteria. Sibbald et al. (1987) showed the some pattern with the heterotrophic
microflagellate Pseudobodo. They were attracted to bacteria in spent medium and
a filtrate ofthe culture, but not to the washed bacteria that were presented without
their associated dissolved cues.
In addition to the ability to sense regions with high food density, avoiding areas
of negative stimuli would be another ecologically advantageous behavior. Many
protozoa orient toward stimuli by changing their swimming pattern to stay in an
area with a positive stimulus, or to move away from a negative or null stimulus
(Fraenkel and Gunn, 1961; Hauser et aI., 1975; Van Houten, 1978). A toxic
bacterium was tested to determine whether the flagellates could avoid a region
which would be disadvantageous to survival. In a test to determine the toxicity
of the bacterium, it was found that movement ceased after 5 min in the suspension
of this bacterium and all flagellate cells died within 20 min. Chromobacterium
elicited a greater apparent chemosensory response (0.97 ± 0.05) from Monas sp.
2 than did Aerobacter, and the response to exudates was again stronger than to
isolated particles. It is possible to speculate that the evolutionary advantage to a
768
BULLETIN OF MARINE SCIENCE, VOL. 43, NO.3, 1988
B.
24
hours
8000
~
A.
90 minutes
~
'0 ~ ~
0.75
~
0.50
~ ~
cEw
6000
.c
'"
~
~
4000
]'j
2000
0
f-
-l?1I
o
1l
"iii
"iii
1.00
0.25
0,00
Bacterial Exudates
Bacleria resuspended
in sterile water
Bacteria in culture
media
Bacteria
in culture
media
Control arm
with sterile
water
Entry arm
with sierile
water
Figure 2. A. Chemosensory index for the phagotrophic phytoflagellate Poterioochromonas malhamensis when tested for 90 min with Aerobacter aerogenes cultures. There was no significant response
during this time period. B. Chemosensory index for Poterioochromonas malhamensis after 24 h when
tested with cultures of Aerobacter aerogenes. These algae exhibited a negative chemosensory response.
Error bars represent standard deviation, N = 3.
bacterial species capable of attracting and killing flagellates is two-fold: to avoid
predation and to utilize killed flagellates as a concentrated source of organic matter
for growth. However, the apparent attractiveness of this toxic bacteria may have
been an artifact of the testing procedure. The density of Chromobacterium in the
T-maze assay, and presumably the exudate concentration, was greater than that
found in nature. Aagellates were apparently killed almost immediately upon
entering the Chromobacterium test arm. Therefore they may have accumulated
there due to immobilization rather than attraction. The experiment does indicate,
however, that Chromobacterium did not give any negative chemical cues detectable to this flagellate species.
A final comparison was to check if a pigmented phagotrophic flagellate would
demonstrate chemosensory responses to a potential particulate food source. Poterioochromonas is known for its ability to utilize a variety of trophic modes
including phagocytosis, osmotrophy, and photosynthesis (reviewed by Sanders
and Porter, 1988). We hypothesized that this species would respond to bacterial
cues in a manner similar to the Monas species. However, Poterioochromonas
demonstrated no response after 90 min to the bacterial cells, exudates, or whole
cultures (Fig. 2). After 24 h, it exhibited a negative chemosensory response to
whole bacterial cultures (Fig. 2). Poterioochromonas may have avoided a region
of extremely high bacterial density to reduce competition, since algae and bacteria
may compete for some of the same nutrients (Currie and Kalff, 1984). Alternatively, the bacteria may have given off allelopathic metabolites which elicited a
negative response from the algae.
These data indicate that flagellates are not only capable of detecting chemicals
in their environment, but that they are effective at accumulating in regions of
high concentrations of prey stimuli. This is an important ability in a coarsegrained aquatic regime which is fairly dilute, but contains patches of high bacterial
biomass and production. On the mesoscale level of a eutrophic lake, flagellates
and bacterial abundances are highest at the metalimnetic boundary and their
numbers are correlated seasonally (Bennett et aI., in prep.). The metalimnetic
plate is a region of high production for both bacteria and phytoplankton and
attracts an assemblage of larger plankters (Bird and Kalff, 1987; McDonough
et aI., 1986; Pick et aI., 1984). The importance of patchiness on a micro scale level
is currently being investigated and debated (Azam and Ammerman, 1984; Biddanda, 1985; Caron et aI., 1982). If micropatches exist long enough for a protist
BENNETT ET AL.: CHEMOSENSORY
RESPONSES OF FLAGELLATED
PROTISTS
769
to sense and adjust its movement pattern toward them, then the chances of it
surviving would be higher compared to a randomly moving flagellate. Jackson
(1987) points out the importance of considering the high degree of heterogeneity
in microbial systems. When taking an average density of nutrients, phytoplankton,
or bacteria per liter, one may get a biased estimate of nutrients or prey items
available in the microenvironment of a flagellated protozoan. In some systems,
a protist could starve in the dilute regions ifit did not have the ability to accumulate
in the patchy areas of high density food.
There is conflicting evidence in the literature on the mechanism that allows
flagellated protists to effectively track concentrations of their prey items. In a
model of bacterial chemosensory responses, Jackson (1987) found that an organism must be small relative to the size of its prey if chemical cues are to be
important. There are physical restraints in diffusion rates of chemicals in the water
from a small «2 ~m) point source, such as bacteria. He states that flagellate
predators are too large relative to the size of their picoplankton prey to utilize
chemosensing abilities, so that they must rely on "touching" (Jackson, 1987). In
this study Monas sp. accumulated in areas with washed bacterial particles preferentially over control regions, which suggests that tactile cues may have been
used for this flagellate species. Flagellates are also known to ingest inert particles,
such as fluorescent polystyrene beads and paint particles, which present only tactile
cues (Bird and Kalff, 1986; McManus and Fuhrman, 1986; Porter, 1988). However, Sibbald et ai. (1987) found no significant migration of Pseudobodo toward
washed bacterial cells in aT-maze after 60 min and concluded that mechanoreception was not involved in the response. Flagellates accumulate in high
concentrations of meat extract (Fox, 1921), amino acids, or bacterial exudates
(Sibbald et aI., 1987; this study) and other dissolved chemical cues (Hauser et aI.,
1975; Spero, 1985), which would rely solely on chemoreception. Responses to
the chemical versus tactile portion of the stimuli may also be species-specific.
These contradictory data suggest that further study with different sizes and species
of flagellates, in different aquatic systems, is necessary to isolate the mechanism
allowing for sensory reception of their prey.
Evidence for flagellate chemosensory abilities has been in the literature for
almost a century (Jennings, 1906; Levandowsky and Hauser, 1978; Van Houten
et aI., 1981). Future investigations of chemosensory responses by microflagellates
should focus on environmentally realistic cues in a physically dynamic aquatic
regime. Concentrated bacterial cultures and amino acids are both unnatural cues
of a food source (Van Houten et aI., 1981), and give suggestive information.
Flagellates should be tested with naturally occurring concentrations and assemblages of bacteria to evaluate the true ecological role of chemosensory behavior
in freshwater micro flagellates. Experiments should be designed to conclusively
show whether mechanoreception, chemoreception or both are involved in the
response. Finally, it is important to test whether this is a chemotactic or a chemokinetic response. This mechanism may not be universal for all flagellated
protists, and will affect the overall timing and efficiency of the response. If a
natural flagellate assemblage were isolated and experiments devised to assess their
sensory abilities in a realistic aquatic regime, then definitive conclusions could
be drawn as to the ecological significance and relative role this ability plays in
the survival strategies of this functional group.
ACKNOWLEDGMENTS
Supported by NSF grant BSR8407928 to K. G. Porter. This is contribution No. 34 to the Lake
Oglethorpe Limnological Association. Discussions with W. K. Fitt and W. J. Wiebe were helpful in
770
BULLETIN OF MARINE SCIENCE, VOL. 43, NO.3, \988
design, analysis, and presentation of these experiments. We acknowledge Skidaway Institute of Oceanography for funding the Zooplankton Behavior Symposium.
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DATEACCEPTED: April 12, 1988.
ADDRESS: Department of Zoology. University of Georgia. Athens. Georgia 30602: PRESENTADDRESS:
Department of Biology, University of North Carolina. Chapel Hill, North Carolina 27599-3280.