GRAZING AND GROWTH OF THE MIXOTROPHIC CHRYSOMONAD POTERIOOCHROMONAS MALHAMENS

Prévia do material em texto

738
 
J. Phycol.
 
 
 
37,
 
 738–743 (2001)
 
GRAZING AND GROWTH OF THE MIXOTROPHIC CHRYSOMONAD 
 
POTERIOOCHROMONAS 
MALHAMENSIS
 
 (CHRYSOPHYCEAE) FEEDING ON ALGAE
 
1
 
Xiaoming Zhang
 
2
 
 and Makoto M. Watanabe
 
National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-0053, Japan
 
The growth and grazing characteristics of 
 
Poterioo-
 
chromonas malhamensis
 
 (Pringsheim) Peterfi (
 
�
 
 
 
Ochromo-
nas malhamensis
 
 Pringsheim) (ca. 8 
 
�
 
m) feeding on phy-
toplankton, including the cyanobacteria 
 
Synechococcus
 
 sp.
(ca. 2 
 
�
 
m) and 
 
Microcystis viridis
 
 (A. Brown) Lemmer-
 
mann (ca. 6 
 
�
 
m) and the green alga 
 
Chlorella pyrenoi-
dosa
 
 Chick (ca. 13 
 
�
 
m), were investigated in laboratory
experiments involving the following treatments: (1)
light without added algal prey (autotrophy), (2) light
with added algal prey (mixotrophy), and (3) dark
with added algal prey (phagotrophy). There were sig-
nificantly higher cell numbers under mixotrophic and
phagotrophic growth than under autotrophic growth.
With phytoplankton as food, growth rates under both
mixotrophy and phagotrophy were about two or three
times higher than those under autotrophy, indicating
that the algal diets were readily able to support the
population growth of 
 
P. malhamensis.
 
 There were no
significant differences in growth rate between mix-
otrophic and phagotrophic cultures during exponen-
tial growth. The ingestion rate of 
 
P. malhamensis
 
 with
algal prey was also similar under both continuous light
and dark. 
 
Poterioochromonas malhamensis
 
 ingested on
 
average 0.27 
 
M. viridis
 
 cells
 
�
 
flagellate
 
�
 
1
 
�
 
h
 
�
 
1
 
 and 0.18
 
C. pyrenoidosa
 
 cells
 
�
 
flagellate
 
�
 
1
 
�
 
h
 
�
 
1
 
 in continuous light
and 0.25 
 
M. viridis
 
 cells
 
�
 
flagellate
 
�
 
1
 
�
 
h
 
�
 
1
 
 and 0.18 
 
C.
 
pyrenoidosa
 
 cells
 
�
 
flagellate
 
�
 
1
 
�
 
h
 
�
 
1
 
 in continuous dark
during exponential growth. The results showed that
light had no effect on the growth and ingestion rates
of 
 
P. malhamensis
 
 for phagotrophy during exponential
growth. However, phagotrophic populations of 
 
P. mal-
hamensis
 
 were incapable of growth in continuous dark-
ness for longer than 5 days. Populations of 
 
P. malha-
mensis
 
 showed no increase when prey was added again
after 4 days in continuous darkness, indicating that
light is necessary for sustained phagotrophic growth of
 
P. malhamensis.
 
 The study suggests that 
 
P. malhamensis
 
,
which has strong tolerance for light, is light dependent
for phagotrophy.
 
Key index words:
 
Chrysomonad; growth rate; herbivo-
 
rous grazer; ingestion rate; mixotrophy; phagotrophy
 
Mixotrophic chrysomonads, which are capable of
both photosynthesis and phagotrophy, are now recog-
nized to play an important role in grazing bacteria and
in regenerating nutrients within marine and freshwa-
ter planktonic ecosystems (e.g. Estep et al. 1986, Bird
and Kalff 1986, 1987, 1989, Boraas et al. 1988, Porter
1988, Salonen and Jokinen 1988, Andersson et al.
1989, Sanders et al. 1989, 1990, Sanders 1991, Bennett
et al. 1990, McKenzie et al. 1995). Previous laboratory
experiments suggested that bacterivory by mixotro-
phic chrysomonads seems to be the dominant mode
of nutrition in all cultures containing heat-killed bac-
teria (Andersson et al. 1989, Caron et al. 1990, Sanders
et al. 1990). Many field studies have also shown the im-
pact of mixotrophic chrysomonads on bacterioplank-
ton in oceans and fresh waters and support the hypoth-
esis that mixotrophic chrysomonads can control the
abundance and growth of bacteria (Bird and Kalff
1986, 1987, Sanders and Porter 1988, Sanders et al.
1989, McKenzie et al. 1995). Therefore, mixotrophic
chrysomonads are generally considered an important
group of bacterivores in some marine and freshwater
planktonic ecosystems. Nevertheless, it has also been
shown that mixotrophic chrysomonads such as 
 
Ochromo-
nas
 
, 
 
Poterioochromonas
 
, and 
 
Chrysamoeba
 
 also feed on vari-
ous algae and bacteria (Pringsheim 1952, Daley et al.
1973, Cole and Wynne 1974, Boraas et al. 1988, Zhang et
al. 1996, Holen 1999), so mixotrophic chrysomonads
are not just functional bacterivores. Despite previous
studies showing the importance of assessing the impact
of grazing on various algae by these pigmented chry-
somonads, very little attention has been given to the eco-
logical role of mixotrophic chrysomonads as herbivores.
There is a clear need to examine the growth and inges-
tion rates of these mixotrophic chrysomonads when sup-
plied with phytoplankton prey.
It has been reported that 
 
Poterioochromonas malha-
mensis
 
, a well-known bacterivorous chrysomonad, de-
rives only 7% of its total carbon budget from photo-
synthesis, the remainder being obtained from bacteria
(Sanders et al. 1990). This alga has been demonstrated
to digest and assimilate carbon and nutrients from bac-
teria with efficiencies similar to truly heterotrophic
nanoflagellates (Caron et al. 1990). In a previous study,
we examined in detail a range of prey grazed by 
 
P. mal-
hamensis
 
 (Zhang et al. 1996). The results indicated that
 
P. malhamensis
 
 is capable of ingesting various kinds of
organisms, ranging from pico-, nano-, and microplank-
ton to motile algae, including the cyanobacteria 
 
Synecho-
coccus
 
 and 
 
Microcystis
 
, the diatom 
 
Achnanthes
 
, the chry-
somonad 
 
Uroglena
 
, and the green algae 
 
Chlorella
 
 and
 
Carteria.
 
 Our study demonstrated for the first time that
 
P. malhamensis
 
 is capable of grazing algae two to three
times larger than its own cell diameter and increasing
in size by 10–30 times that of a normal cell. Thin sec-
 
1
 
Received 30 August 2000. Accepted 6 June 2001.
 
2
 
Author for correspondence: e-mail Zhang.xiaoming@nies.go.jp.
 
GRAZING AND GROWTH OF 
 
P. MALHAMENSIS
 
739
 
tions examined by TEM showed that 
 
P. malhamensis
 
was not only ingesting but also digesting the prey 
 
Syn-
echococcus
 
, 
 
Microcystis
 
, 
 
Chlorella
 
, and 
 
Chlamydomonas
 
 (Zhang
et al. 1996). These studies therefore suggested that 
 
P. mal-
hamensis
 
 may be an important grazer not only of bacteria
but also of pico-, nano-, and microphytoplankton. Despite
the potential importance of its effect on phytoplankton, it
remains uncertain whether 
 
P. malhamensis
 
 can use such
phytoplankton to support its population growth.
Here we present further work that compares the in-
gestion and growth rates of 
 
P. malhamensis
 
 feeding on al-
gae during the day and night. We selected three species
of alga differing in size, shape, and taxonomy, the cy-
anobacteria 
 
Synechococcus
 
 sp. (2 
 
�
 
m) and 
 
Microcystis viri-
dis
 
 (ca. 6 
 
�
 
m) and the green alga 
 
Chlorella pyrenoidosa
 
 (ca.
13 
 
�
 
m), for grazing experiments. Our results confirm
that not only bacteria but also phytoplankton algae could
support the population growth of 
 
P. malhamensis.
 
materials and methods
 
Algal culture.
 
The culture strain of 
 
P. malhamensis
 
 (ca. 8 
 
�
 
m
diameter, 268 
 
�
 
m
 
3
 
 volume) was from the chrysomonad collec-
tion of the first author (X. Z.) and is maintained at the Microbial
Culture Collection, National Institute for Environment Studies,
Japan Environment Agency. Cultures were grown in 2-L Erlen-
meyer flasks with 1 L modified AF-6 medium (Kato 1982) under il-
lumination of ca. 100 
 
�
 
mol photons
 
�
 
m
 
�
 
2
 
�
 
s
 
�
 
1
 
 with a photoperiod
of 12:12-h light:dark from daylight fluorescent lamps at 20
 
�
 
 C.
 
Preparation of prey algae.
 
Cultures of thecyanobacteria 
 
Syn-
echococcus
 
 sp. (axenic, clonal, PS-717) and 
 
M. viridis
 
 (axenic,
clonal, NIES-102) and the chlorophyte 
 
C. pyrenoidosa
 
 (axenic,
clonal, NIES-226), used as prey organisms, were obtained from
the Microbial Culture Collection, National Institute for Environ-
ment Studies, Japan Environment Agency. The algal strains were
grown in 200-mL Erlenmeyer flasks with 100 mL of growth me-
dium suited for each algal culture (Watanabe and Nozaki 1994).
Each algal species was grown to a moderately dense culture, har-
vested by centrifugation, and heat killed at 70
 
�
 
 C for 30 min.
 
Experimental treatments.
 
The responses of growth and inges-
tion rates to prey availability were determined for each prey—
 
Synechococcus
 
 sp, 
 
M. viridis
 
, and 
 
C. pyrenoidosa
 
—or a mixture of
25% of each of them and a bacterium. The final concentration
of prey was adjusted to 6 
 
�
 
 10
 
6
 
 cells
 
�
 
mL
 
�
 
1
 
. For the feeding ex-
periments, 
 
P. malhamensis
 
 cells in exponentially growing popu-
lations were harvested and washed three times with sterile me-
dium. The prey algae were added to 
 
P. malhamensis
 
 cultures
and incubated for 10 days. Experiments with prey were divided
into two parts, with or without light. Cultures with prey incu-
bated in continuous light were under illumination of ca. 100
 
�
 
mol photons
 
�
 
m
 
�
 
2
 
�
 
s
 
�
 
1
 
. Cultures with prey incubated in contin-
uous dark were wrapped in foil and sampled in almost total
darkness. Control cultures in the light without prey were incu-
bated in parallel with other cultures. All experimental treat-
ments were performed in three replicate cultures. The growth
temperature for all cultures used was 20
 
�
 
 C. Samples were ob-
served daily, and all samples for cell counts were preserved with
buffered glutaraldehyde (1% final concentration).
 
Determination of growth rate.
 
Cell counts from triplicate sam-
ples were determined using hemacytometer chambers through-
out the growth cycle of the population. Samples for prey and algal
cell counts were taken aseptically at 
 
t
 
 
 
�
 
 0 and at about 24-h inter-
vals up to day 8 in each experimental treatment. Specific growth
rates of P. malhamensis, �(d�1), in the various treatments were de-
termined from regressions of the linear portion of the growth
curves expressed as the natural log of the population density ver-
sus time (i.e. exponential growth phase). The specific growth rate
of P. malhamensis, �(d�1), was calculated as �(d�1) � (lnPt �
lnP0)/t, where P0 is the initial concentration of P. malhamensis and
Pt is the final concentration of P. malhamensis (cells�mL�1) after
time t (d).
Ingestion rate. Ingestion rates of algae by P. malhamensis were
calculated using the equations of Heinbokel (1978). Ingestion
rates were determined by the disappearance of prey in the cul-
tures using the following formula: Ic � �P/F�t � �P/[(F2 �
F1) 	 (lnF2 � lnF1) � �t], in which �P is the decrease in prey
cell number during time interval �t and F2 and F1 are the pred-
ator cell densities (cells�mL�1) at the end and beginning, re-
spectively, of each interval (Heinbokel 1978).
Although bacteria may have been present in the experimen-
tal cultures, the collected P. malhamensis cells were washed
three times with sterile medium to decrease bacterial numbers
before the experiments. Therefore, ingestion and growth rates
were analyzed only in relation to prey algal density.
results
Growth rates. Populations of P. malhamensis fed on
M. viridis exhibited a 1-day lag phase followed by expo-
nential growth for incubation periods as long as 3 days
under continuous light or dark (Fig. 1, A and B). Dur-
ing exponential growth, there was not a significant dif-
ference in growth rate between cells grown with M. vir-
idis as prey under continuous light and dark (Fig. 1, A
and B, Table 1; Student’s t-test, P 
 0.05). In the con-
trol experiment, cell numbers of P. malhamensis in-
creased slowly (Fig. 1C). However, the cell numbers of
P. malhamensis for phagotrophy in cultures placed in
continuous dark using M. viridis as prey increased up
to day 4 after inoculation. Then, the cell densities of P.
malhamensis decreased gradually after day 4, whereas
M. viridis prey cells increased slightly in the cultures
(Fig. 1B). The same pattern was observed for P. malha-
mensis ingestion of C. pyrenoidosa, except that the pop-
ulation of P. malhamensis showed constant exponen-
tial growth for 4 days without a lag phase under both
light and dark (Fig. 2, A and B). There was no signifi-
cant difference in growth rate between cells grown
with C. pyrenoidosa as prey under continuous light and
dark (Table 1; t-test, P 
 0.05). There were major con-
trasts in the patterns of growth in the two grazing ex-
periments with or without light, which coincided with
the end of exponential growth of P. malhamensis in
both mixotrophic and phagotrophic experiments. In
mixotrophic experiments, there was a continuous in-
crease in the cell numbers of P. malhamensis after the
end of exponential growth. Populations of P. malha-
mensis fed on M. viridis or C. pyrenoidosa exhibited sta-
tionary phase growth by day 4 in continuous light
(Figs. 1A and 2A). In phagotrophic experiments, the
cell numbers of P. malhamensis decreased gradually af-
ter the end of exponential growth, and no stationary
phase growth was observed (Figs. 1B and 2B). The cell
yields grown with the same prey were significantly
higher in all light treatments (mixotrophy) than in all
dark ones (phagotrophy) (Table 1; t-test, P � 0.05).
There were higher rates of increased cell numbers
under phagotrophic or mixotrophic growth than un-
der autotrophic growth. With both M. viridis and C.
pyrenoidosa as food prey, growth rates in the light or
dark were about two or three times higher than those
without added food prey (autotrophic) (Figs. 1 and 2,
740 XIAOMING ZHANG AND MAKOTO M. WATANABE
Fig. 1. Growth of Poterioochromonas malhamensis (�, �)
feeding on Microcystis viridis at a prey concentration of 6 � 106
cells�mL�1 and changes in cell density of prey (�, �) as a func-
tion of incubation time under continuous light (A) or continu-
ous dark (B). A control experiment (C) under continuous light
showing autotrophic growth of P. malhamensis (�). Vertical
bars show SD (n � 9, based on three replicate cultures).
Table 1). The growth rates between autotrophic
growth and phagotrophic or mixotrophic growth
were significantly different in all treatments (Table 1;
t-test, P � 0.05). Addition of prey had a significant ef-
fect on growth rate (analysis of variance [ANOVA], F �
109, P � 0.0001). For the mixotrophic (light with
prey) experiment, the growth rate (d�1) of P. malha-
mensis was 0.71 for Synechococcus sp. as food and 0.85
with M. viridis, 0.75 for C. pyrenoidosa, and 0.89 when a
mixture of bacteria, Synechococcus sp., M. viridis, and C.
pyrenoidosa was added as food (Table 1). For the phag-
otrophic (dark with prey) experiments, the growth
rate (d�1) was 0.81 with Synechococcus sp. as food, 0.90
with M. viridis, 0.79 with C. pyrenoidosa, and 0.82 for a
mixture of bacteria, Synechococcus sp., M. viridis, and C.
pyrenoidosa as food (Table 1). There was no significant
difference in growth rate between mixotrophy and
phagotrophy with the same prey for all treatments
(t -test, P 
 0.05); also no significant difference was
seen in growth rate between mixotrophy and phag-
otrophy with different prey (P 
 0.05). Light or dark
had no significant effect on growth rate (ANOVA, F �
1.715, P � 0.209).
The effect of light and prey density was studied in
an experiment where M. viridis was added again to P.
Fig. 2. Growth of Poterioochromonas malhamensis (�, �)
feeding on Chlorella pyrenoidosa at a prey concentration of 6 �
106 cells�mL�1 and changes in cell density of prey (	, 
) as a
function of incubation time under continuous light (A) or con-
tinuousdark (B). Vertical bars show SD (n � 9, based on three
replicate cultures). A control experiment (C) under continu-
ous light showing autotrophic growth of P. malhamensis (�).
GRAZING AND GROWTH OF P. MALHAMENSIS 741
malhamensis cultured in continuous dark with M. viri-
dis. A population of M. viridis as high as 9.8 � 106
cells�mL�1 was added to cultures of P. malhamensis.
The original dark sample was split into two cultures
on day 4 after adding the prey. One culture was main-
tained in the dark and the other transferred to the
light (Fig. 3). After 24 h, there was an obvious in-
crease in P. malhamensis cell density in the light and a
decrease in the dark (Fig. 3). The cell numbers in the
light treatment were significantly higher than in the
dark when tested using a two-sample t-test for adding
prey at 24 h and 48 h (P � 0.05, Table 2).
Ingestion rate. The maximum ingestion rate occurred
during the first sampling interval during which the prey
density decreased precipitously. When M. viridis was used
as food prey, ingestion rates were 3.2 cells� flagellate�1�h�1
in the light and 3.9 cells�flagellate�1�h�1 in the dark (Ta-
ble 3). The maximum ingestion rate was not significantly
different between the light and dark conditions with
M. viridis as prey (t-test, P 
 0.05). When C. pyrenoi-
dosa was used as food prey, the ingestion rate was
1.8 cells�flagellate�1�h�1 in the light and 1.2 cells�
flagellate�1�h�1 in the dark (Table 3). There was not a
significant difference in maximum ingestion rate be-
tween the light and dark treatments when C. pyrenoi-
dosa was used as prey (P 
 0.05). However, prey type
had a significant effect on maximum ingestion rates
(ANOVA, F � 33.8, P � 0.0002).
During exponential growth, the average ingestion
rate of P. malhamensis for M. viridis was 0.27 cells�
flagellate�1�h�1 in continuous light and 0.25 cells�
flagellate�1�h�1 in continuous dark (Table 3). On the
other hand, the average ingestion rate of P. malhamen-
sis for C. pyrenoidosa was 0.18 cells�flagellate�1�h�1 in
continuous light and 0.18 cells�flagellate�1�h�1 in the
dark. There was not a significant difference in average
ingestion rate between the light and dark treatments
when M. viridis or C. pyrenoidosa was used as prey (P 
0.05). However, prey type had a significant effect on
ingestion rates (ANOVA, F � 34.9, P � 0.0001).
discussion
Little direct experimental evidence of mixotrophic
chrysomonads growing on algae has been documented
until now (Holen 1999). Although Cole and Wynne
(1974) also showed that mixotrophic Ochromonas danica
Prings is capable of grazing the toxic cyanobacterium
Table 1. Growth parameters for Poterioochromonas malhamensis cultured under autotrophic, mixotrophic, and phagotrophic conditions.
Prey Size (�m) Treatment Growth rate �(d�1) Cell yield (cells�mL�1)
Synechococcus sp. 2.0 Light 0.71 � 0.04a 3.76 � 105 � 0.13a
Dark 0.81 � 0.087a 1.01 � 105 � 0.13b
Microcystis viridis 5.6 Light 0.85 � 0.097a 8.45 � 105 � 1.03a
Dark 0.90 � 0.058a 4.27 � 105 � 0.45b
Chlorella pyrenoidosa 13.3 Light 0.75 � 0.04a 1.29 � 106 � 0.16a
Dark 0.79 � 0.03a 4.86 � 105 � 0.56b
B
S.
M.
C. Light 0.89 � 0.04a 9.90 � 105 � 0.30a
Dark 0.82 � 0.04a 2.41 � 105 � 0.29b
Autotrophy Light 0.32 � 0.031b
Means � SD (n � 3, based on three replicate cultures) are not significantly different in growth rate between the light and dark
treatments with the same prey (common letters) (P 
 0.05), not significantly different between the light and dark treatments with
different prey (P 
 0.05), but significantly different between autotrophy and mixotrophy or phagotrophy (different letters) (P � 0.05).
Cell yield was calculated as the maximum algal density minus the initial density. Means � SD (n � 3, based on three replicate cultures)
are significantly different between the light and dark treatments with the same prey (different letters) (P � 0.05). B, bacteria; C,
Chlorella pyrenoidosa; M, Microcystis viridis; S, Synechococcus sp.
Fig. 3. Effect of light and prey on growth of Poterioochromo-
nas malhamensis. Poterioochromonas malhamensis was fed Microcys-
tis viridis at an initial concentration of 5.6 � 106 cells�mL�1 and
incubated in continuous dark. After 4 days, the dark-treated
cultures received M. viridis as prey again at an initial concentra-
tion of 9.8 � 106 cells�mL�1 and split into two parts (arrow),
one of which was maintained in continuous dark (�) and the
other incubated in continuous light (�). Vertical bars show SD
(n � 8–12, based on three replicate cultures).
Table 2. Poterioochromonas malhamensis comparison of cell
numbers after adding Microcystis viridis as prey.
Time Treatment
Cell numbers
(�105�mL�1)
24 h Light 5.38 � 0.68
Dark 3.48 � 0.57
48 h Light 11.57 � 1.01
Dark 2.87 � 0.31
Means � SD (n � 8, based on three replicate cultures) are
significantly different between the light and dark treatments
(P � 0.05).
742 XIAOMING ZHANG AND MAKOTO M. WATANABE
Microcystis aeruginosa, the grazing and growth rates of
O. danica on M. aeruginosa were not given. The present
study is apparently the first to provide data on both graz-
ing and growth rates of a mixotrophic chrysomonad feed-
ing on algae. Our results indicate that the mixotrophic P.
malhamensis is capable of ingesting three kinds of algae
to support its growth. The grazing and growth charac-
teristics of P. malhamensis indicate that it is well suited
for existence in phytoplankton communities. When
feeding on algae, P. malhamensis showed growth rates
for both mixotrophy and phagotrophy two to three
times higher than those for autotrophy, indicating
that the algal diets were readily able to support the
population growth of P. malhamensis. However, the
growth rates of herbivorous P. malhamensis were lower
in this study (0.71–0.90 d�1) than in earlier ones (1.5–
2.2 d�1), when bacteria as prey were not limited
(Sanders et al. 1990, Caron et al. 1990). There are two
possible explanations for this difference. First, the
prey density in our study may have been below the sat-
urating concentrations for sustaining the maximum
specific growth rates of P. malhamensis. Sanders et al.
(1990) also pointed out that prey density was the pri-
mary factor influencing the ingestion and growth
rates of P. malhamensis. Second, different strains of the
same species collected from different places may have
different growth rates, as we observed in two strains of
P. malhamensis showing different growth rates (unpub-
lished data).
The maximum ingestion rates (about 1.2–3.9 cells�
flagellate�1�h�1) calculated for P. malhamensis in this
study were lower than rates (about 89–103 bacte-
ria�flagellate�1�h�1) previously determined for the
same species feeding on bacteria (Sanders et al. 1990,
Caron et al. 1990). The lower rates can be explained
by the considerably larger cell size (6–13 �m) of the
prey used in our study. For example, when M. viridis (ca.
6 �m) was used as food, 3.9 cells�flagellate�1�h�1 in the
light and 3.2 cells�flagellate�1�h�1 in the dark were de-
termined, whereas only 1.8 cells�flagellate�1�h�1 in the
light and 1.2 cells�flagellate�1�h�1 in the dark were mea-
sured when the larger sized C. pyrenoidosa (ca. 13 �m)
was used as food. Our results indicate that prey type had
a significant effect on maximum ingestion rates
(ANOVA, F � 33.8, P � 0.0002).
Mixotrophy is commonly found among chrysomonads
and other nanoplankton (Bird and Kalff 1986, Sanders et
al. 1989, 2000, Sanders 1991). Mixotrophic chrysomonads
such as Ochromonas, Dinobryon, and Uroglena can occur at
very high densities in natural environments (Kimura and
Ishida 1985, 1986, Bird and Kalff 1986, Sanders et al.
1989, Olrik and Nauwerck 1993). Previous studies
have reported that mixotrophic Ochromonas is capable
of grazing cyanobacteria (Anacystis and Microcystis) and
a green alga (Daley et al. 1973, Cole and Wynne 1974,
Boraas et al. 1988). Ochromonas sp. was found to signif-
icantly reduce a bloom of small unicellular centric di-
atoms in Hjarbak Fjord Lake, Denmark (Olrik andNauwerck 1993). We have also observed that Poterioo-
chromonas spp. were present during toxic blooms of the
cyanobacterium Microcystis in lakes of Thailand and P. R.
China (unpublished data). Therefore, herbivory may be
a common behavior among mixotrophic chrysomonads.
Because of their ability to graze a wide size range of prey,
mixotrophic chrysomonads may be effective competi-
tors with other grazers for pico- and nanoplanktonic al-
gae other than bacteria and may make a stronger link
transferring energy of phytoplanktonic algae and bacte-
ria to nanoplankton grazers by increasing trophic effi-
ciency. Our observations and those in the literature im-
ply that mixotrophic chrysomonads can influence not
only the population dynamics and community composi-
tion of bacteria, but also pico- and nanoplankton algae.
Various factors influence both the ingestion and
growth rates of mixotrophic chrysomonads, including
prey density, light, pH, inorganic nutrients, and dis-
solved organic carbon (Bird and Kalff 1987, 1989,
Andersson et al. 1989, Sanders et al. 1990, Caron et al.
1990, 1993, Holen 1999); among these light has been
studied in most detail. Light had a marked effect on
the ingestion rate and growth of Dinobryon cylindricum
(Caron et al. 1993). Dinobryon cylindricum cultured at a
light intensity � 150 �mol photons�m�2�s�1 had inges-
tion rates of approximately 5–10 bacteria�flagellate�1�h�1
during the exponential growth phase; however, when
cultured in the dark D. cylindricum rapidly declined in
density and ceased ingesting bacteria (Caron et al.
1993). We also observed that another species of Dino-
bryon, D. sociale, and Uroglena americana were incapable
of growth and ingestion in continuous dark with bac-
teria as prey (unpublished data). The presence or ab-
sence of light, in contrast, had no direct effect on phago-
trophy for Ochromonas sp. or P. malhamensis (Andersson
et al. 1989, Caron et al. 1990, Sanders et al. 1990, Sib-
bald and Albright 1991). The ingestion and growth
rates in both light and dark were the same when bac-
teria were added as prey (Andersson et al. 1989, Caron
et al. 1990, Sanders et al. 1990).
In the present study there was no significant differ-
ence in grazing and growth rates between phagotro-
phy and mixotrophy during the exponential growth
phase, indicating that light had no effect on P. malha-
mensis during this period. This appears similar to ob-
servations in previous studies on the same species and
on Ochromonas sp. (Andersson et al. 1989, Caron et al.
Table 3. Poterioochromonas malhamensis comparisons of
maximum ingestion rate (cells�flag.�1�h�1) and averaged
ingestion rate (cells�flag.�1�h�1, during exponential growth
phase) for mixotrophic (light) and phagotrophic (dark) cells.
Prey (�m) Treatment
Max. ingestion 
rate
Ingestion 
rate
Microcystis viridis (5.6) Light 3.2 � 0.4 0.27 � 0.01
Dark 3.9 � 0.8 0.25 � 0.04
Chlorella pyrenoidosa (13.3) Light 1.8 � 0.5 0.18 � 0.02
Dark 1.2 � 0.4 0.18 � 0.01
Means � SD (n � 3, based on three replicate cultures) are not
significantly different between the light and dark treatments
with the same prey (P 
 0.05) but are significantly different
between two kinds of prey (P � 0.05).
GRAZING AND GROWTH OF P. MALHAMENSIS 743
1990, Sanders et al. 1990). However, our populations
of P. malhamensis were incapable of growth and inges-
tion in continuous dark for a period longer than 5
days with M. viridis or C. pyrenoidosa. During the sta-
tionary phase, cell numbers of P. malhamensis were
nearly constant, whereas decrease in prey density was
observed. In contrast, there was a gradual decrease in
cell density of P. malhamensis and no change or a
slight increase in prey density in continuous dark. We
confirmed that light is an important factor affecting
phagotrophy of P. malhamensis by adding prey again
on day 4 for a phagotrophic experiment (Fig. 3). In
this case, the increase in density of the predator was
probably due not only to the prey but also the light,
because the population of P. malhamensis kept in con-
tinuous dark had not yet increased after the addition
of a high density of prey. Five days in the dark may
have been the tolerance threshold of P. malhamensis
in our study. It is therefore concluded that although
P. malhamensis is light tolerant, it is also dependent on
light for sustained phagotrophy. This conclusion dif-
fers from those of previous studies (Andersson et al.
1989, Caron et al. 1990, Sanders et al. 1990). All cul-
tures for determining ingestion and growth rates in
our study were incubated for more than 8 days,
whereas ingestion rates were measured during the
first sampling interval by Caron et al. (1990) and dur-
ing 52–70 hours by Sanders et al. (1990). Based on
the above results, it is suggested that the measure-
ments by Caron et al. and Sanders et al. were deter-
mined within the tolerance threshold for light. Light
tolerance of mixotrophic chrysomonads may vary with
species. Dinobryon cylindricum, D. sociale, and U. americana
are very light sensitive (Caron et al. 1993, our unpub-
lished data), whereas Ochromonas and Poterioochromonas
have a strong tolerance for light (Andersson et al. 1989,
Caron et al. 1990, Sanders et al. 1990, this study). The
present study agrees with the hypothesis that there is a
requirement for some factor(s) supplied by photosyn-
thesis in mixotrophic chrysomonads for phagotrophy
(Caron et al. 1993).
X. Z. was supported by a National Institute Postdoctoral Fellowship
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