Chemical Induction Of Colony Formation In A Green Alga

Limnol. Oceanogr., 39(7), 1994,1543-1550
0 1994, by the American
Society of Limnology
and Oceanography,
Inc.
Chemical induction of colony formation in a green alga
(Scenedesmus acutus) by grazers (Daphnia)
Winfried Lampert, Karl Otto Rothhaupt, and Eric von Elert
Max Planck Institute
for Limnology,
Postfach 165, 24302 PlSn, Germany
Abstract
The green alga, Scenedesmusacutus, grows in culture in unicellular form, but it forms colonies (coenobia)
when exposed for 48 h to a chemical released by the grazer Daphnia magna. The colony-forming
response
can be evoked only in growing cells. The Daphnia factor affects colony size but not algal growth rate.
The minimum concentration of Daphnia factor that induces colony formation is equivalent to a Daphnia
biomass of 0.5 mg dry wt liter-* in the culture medium. Actively feeding daphniids induce a stronger
response than starved ones. Homogenized Scenedesmus, homogenized Daphnia, ammonium, and urea
are not effective. The Daphnia factor is a nonvolatile, organic substance of small molecular mass (< 500
Da). It is moderately lipophilic, heat stable, pH-resistant in a range from 1 to 12, and not affected by
treatment with Pronase E. The chemical activity is not lost when the substance is dried but disappears
during incineration. Colony formation can be interpreted as a grazing defense mechanism. The phenotypic
response may have evolved because of the tradeoff between higher sinking rates and grazing resistance
of colonial forms.
Phytoplankton can be extremely variable, as
species are composed of many different clones
that can replace each other under differing environmental conditions (Wood and Leatham
1992). Clones established from single cells can
also change their morphology when grown under laboratory conditions; thus, they are phenotypically
plastic. The green algal genus
Scenedesmus is known to be notoriously phenotypically flexible (Trainor 199 1). Individual
strains of various Scenedesmus species can
grow as unicells or can form colonies (coenobia) of four or eight cells. The cells can also
vary with respect to the number and size of
spines.
Trainor (1992) claimed that the phenotypic
change in Scenedesmusis an ordered sequence
of ecomorphs that can be defined as cyclomorphosis (sensu Black and Slobodkin 1987)
driven by environmental
factors. Various abiotic factors (nutrients, pH) and the age of the
culture affect colony size (Egan and Trainor
1989), but temperature is particularly effective
in controlling Scenedesmus phenotypes. Unicells are predominant at warm temperatures,
Acknowledgments
We are indebted to Ellen van Donk and Dag Hessen
who stimulated this work and encouraged us to pursue
this problem. We also thank Maren Volquardsen
and
Heinke ClauBen for help with the algal tests, Heinke Buhtz
for cell counts, and Nancy Zehrbach for linguistic corrections of the manuscript.
while colonies dominate at low temperatures
(1 OOC)(Trainor 1993a).
Recently, Hessen and van Donk (1993) discovered that colony formation and spine enforcement in Scenedesmus subspicatus can be
induced by a biotic factor-a dissolved chemical released from Daphnia magna. Under the
influence of a “Daphnia factor,” unicell Scenedesmus formed eight-cell colonies within 3-5
d. Hessen and van Donk (1993) suspected that
large, spine-armored colonies may be protected from grazers, and they were able to demonstrate that Daphnia had lower grazing rates
on colonies compared to single cells.
The study of Hessen and van Donk (1993)
is particularly exciting as it not only demonstrates the phenotypic development of a defensive mechanism in this alga but also shows
that the algal response is mediated by a chemical stimulus released by the grazer (kairomone). Numerous such responses have recently been described for zooplankton (Larsson
and Dodson 1993), and they have stimulated
a growing interest in the theory of phenotypic
plasticity. Phenotypic changes may be of great
importance in phytoplankton-zooplankton
interactions.
These ideas caused us to repeat Hessen and
van Donk’s experiment with a different Scenedesmus species in a more quantitative and statistically rigorous way. Our aim was to test
whether colony formation occurs at concentrations that are realistic for natural zooplank-
1543
1544
Lampert et al.
ton abundances and to determine how the
chemical activity of the Daphnia factor is
modified. The experiments were also designed
to test whether a tradeoff between colony formation and algal growth rate can be detected.
Finally, we performed tests to begin to characterize the chemical nature of the colony-inducing factor.
Material and methods
Scenedesmus acutus Meyen has been cultured in chemostats in our laboratory for many
years (Lampert et al. 1988) with a modified,
purely inorganic Chu 12 medium (Miiller 1972)
at a growth rate of 0.7 d-l. The algal population consists mainly of single cells (mean
equivalent spherical diameter “ESD,” 5.9 pm;
mean volume, 108 pm3). On average, 1.7 cells
form an aggregate and the cells do not have
spines under these culture conditions. The chemostats are not axenic, but bacterial biomass
is negligibly small.
Tests were run in batch cultures in loo-ml
cellulose-plug-stoppered
Erlenmeyer
flasks
containing 50 ml of medium. In a standard
biotest, each flask contained 45 ml of fresh Chu
medium, 3 ml of algal inoculum, and either
another 2 ml of Chu medium (controls) or 2
ml of test water (treatment). The initial algal
concentration was -1.25 x lo5 cells ml-l.
Flasks were incubated in a temperature-controlled (22°C) and illuminated cabinet on a rotating shaking table (80 rpm). Continuous light
from above was provided by fluorescent lamps
(photon supply rate, 250 pmol m-2 s-l). Standard incubation time was 48 h, although this
was occasionally varied for special tests. The
amount of test water was also increased in some
tests, but the algal inoculum was always 3 ml.
All treatments and controls were run in triplicate.
Daphnia magna Straus from a strain maintained in our laboratory for many years was
cultured in 1.5-liter jars in membrane-filtered
lake water from mesotrophic Schiihsee with
Scenedesmusas food. To produce the standard
Daphnia factor, we kept 40 adult daphniids
(total dry mass, - 10 mg) in 200 ml of filtered
lake water with sufficient Scenedesmus food.
Since we did not find differences in the chemical activity of the water after 12, 24, and 36
h of Daphnia incubation, we used 24 h as the
standard treatment. Water from the Daphnia
jar was passed through a 0.1 -pm membrane
filter before it was added to the algal cultures.
The effect of “starved” Daphnia was tested in
some experiments in which we incubated
daphniids as in the standard procedure but did
not feed them.
Initial and final algal densities and particle
size distributions
were measured in a CASY
particle analyzer (l OO-pm capillary). The size
range from 3.5- to 15.5-pm ESD (270 channels) was analyzed. Depending on algal density, we diluted the samples 1 : 10 or 1 : 100
and measured 4 x 0.2 ml or 4 x 0.4 ml to
count between 5,000 and 10,000 particles. A
subsample of each treatment was preserved in
Lugol’s fixative and the number of cells per
colony was determined for - 400 aggregates in
the inverted microscope. Algal growth rates
were calculated from the initial and final samples using both algal volume and cell numbers.
Because the particle counter determines the
number of aggregates rather than the number
of individual cells, we calculated the mean individual cell volume from the mean particle
volume (determined by CASY) and the mean
number of cells per aggregate (determined by
microscopic counts). The total algal volume
(pm3 ml-‘) was then divided by the individual
cell volume (pm3) to calculate the number of
cells ml - l.
We gained some information
about the
chemical nature of the agent by subjecting sterile-filtered Daphnia water to various treatments before using it in the standard test procedure to see whether the chemical activity was
still present. These treatments needed some
time and the biotest could only be started the
next day. A subsample of the untreated, filtered chemically active water was therefore
stored at room temperature for the time the
handling of the treatments required and was
then stored in a refrigerator overnight, as were
the treated samples. This subsample was used
as a positive control to ensure that the agent
did not lose its chemical activity during storage.
Molecular size was determined by ultrafiltration with a YC05 membrane, which retains
molecules >500 Da. Twenty milliliters
of
chemically active water were filtered, and the
membrane was rinsed with another 20 ml of
Chu medium before the membrane was dry.
Just before the membrane was dry again, the
Daphnia
induces algal colonies
supernatant was diluted with 40 ml of Chu
medium. Both filtrate and supernatant were
adjusted to pH 8.
We adjusted 20 ml of chemically active water to pH 1.0 with 25% HCl and heated it to
60°C in a closed vessel for 30 min. After cooling to room temperature, we readjusted the pH
to 8.0 with 2 N NaOH. The same procedure
was carried out at pH 12 and 60°C.
The chemically active water was dried in a
rotating evaporator at 40°C under a vacuum.
The residues were redissolved in the same
amount of ultrapure water. In a separate treatment, the dry residues were heated for 5 min
in the flame of a Bunsen burner to destroy
organic substances.
Lipophily of the substance was tested by solid-phase extraction of chemically active water.
A Cl8 cartridge was rinsed with 10 ml of methanol and conditioned with 50 ml of ultrapure
water. We ran 20 ml of chemically active water
through the cartridge and adjusted the water
passed to pH 8. The cartridge was extracted
with 10 ml of methanol, the solvent was evaporated at 40°C under a vacuum, and the residues were redissolved in 20 ml of Chu medium.
To test for a proteinaceous substance, we
incubated 20 ml of the chemically active water
with 5 ml of 165 mM phosphate buffer (pH
7.5) and 1 ml of Pronase-E solution (Sigma, 2
mg ml-l, 33 mM phosphate buffer, pH 7) for
5 h at room temperature. Following the enzyme treatment, we adjusted samples to pH
8.0 and diluted them to 40 ml. Controls were
prepared in exactly the same way but with 1
ml of water instead of the enzyme.
Results
Colony formation - The addition of filtered
Daphnia water to the Scenedesmus cultures
resulted in a dramatic increase in the number
of cells per colony. For example, 2 ml of water
from a culture of 200 Daphnia liter-’ (final
concn equivalent to 2 mg dry wt liter - l) caused
~50% of the cells to remain in eight-celled
coenobia; only 10% remained single cells. The
opposite was observed in the controls (Fig. 1).
This figure is similar to the results of Hessen
and van Donk (1993) with S. subspicatus, although no spines are induced in our strain of
S. acutus.
1545
0.8 -
s 0.6 g
Z
.E
=
0.4 -
s:
2
o- o 2 _
’
0.0 12345678
Cells / Colony
1.0
g
1
- I’
I’
0.8 - 1’1
I’
0.0
I
0
500
I
I
1000
1500
\
2000
Volume
Fig. 1. Effect of 2 ml of filtrate from a Daphnia culture
(200 liter-‘) added to a 50-ml Scenedesmus culture after
48 h. Different measures for colony formation from the
same experiment. Upper panel-Distribution
of the number of cells forming a single aggregate in controls (white
bars) and treatments (shaded bars). Error bars represent 1
SD (n = 3). Lower panel-Size
distributions
of particles
as determined in the particle counter. Broken line, controls; solid line, treatments. The abscissa represents 270
measurements (channels). Each curve is the mean of three
replicates smoothed by running averages of five.
Cell aggregation is reflected in the distribution of particle sizes (Fig. 1). Small particles,
dominant in the controls, disappeared in the
treatments, while large particles increased.
Control particles were slightly larger (mean,
145 l.cm3) than at the beginning of the incubation (mean, 108 pm3), but treatments were
considerably larger (mean, 356 pm3). Note that
Lampert et al.
1546
Table 1. Multiple-range
ANOVA
for mean particle
volumes (+ 1 SE, n = 3) from Exp. A. Effect of varying
numbers of Daphnia in the incubation water (2 ml added
to 48 ml of algal culture). “2 x ” denotes a second addition
of 2 ml of incubation water after 24 h. Daphnia homogenate is equivalent to 200 liter-l. Overall ANOVA: 1;9,20
= 156.1; P < 0.000 1. Asterisks sharing the same vertical
column indicate treatments that are not significantly different at the 95% level (Tukey’s test).
600
0
500
l
Treatment
Control
Daphnia homogenate
Daphnia liter-l
100
I
1
I
I
2
3
I
I
I
456
Cells per aggregate
Fig. 2. Relationship between the average number of
cells per aggregate (determined microscopically)
and the
mean volume per particle (determined by particle counter)
in treatments of various strengths.
the 270 channels of the particle counter are not
equally spaced on the x-axis when volume is
plotted (distances increase to the right); hence,
mean particle volume cannot be estimated directly from this curve.
Cells per colony were counted for 16 treatments with three replicates each, and the mean
number of cells per colony was compared with
the mean particle volume as determined with
the CASY. Both parameters are highly correlated (Fig. 2). Thus, mean particle volumes
could be used for statistical comparison of the
treatments. The regression of the 48 measurements plotted in Fig. 2 is
log(mean particle volume)
= 2.127 + 0.726 x log(cells aggr.-l)
(r2 = 0.868). The slope is < 1, which indicates
a decreasing individual cell size in treatments
with aggregated cells.
The mechanism of colony formation
is
probably not an aggregation of already existing
single cells. More likely, dividing cells remain
adhered when they leave the parent cells
(Trainor 1993b). Colony formation took place
only in growing algal cultures in full medium.
Daphnia culture water had no effect on nonmultiplying cells in nutrient-poor medium (e.g.
in lake water from Schiihsee without additional nutrients).
0
5
10
50
50(2x)
100
200
200(2x)
Mean particle
volume
Homogeneous
145.3(2.3)
149.7(1.9)
*
*
156.0(4.9)
153.3(4.1)
158.3(4.1)
155.7(0.9)
174.3(4.5)
203.0(5.6)
356.0(8.0)
357.3(16.3)
*
*
*
*
*
groups
*
*
*
*
Algal growth rates -Differing
treatments affected colony size but not algal growth rates
(i.e. the Daphnia factor controls only the adhesion of cells, not their production). Growth
rates calculated from total algal volume were
higher than growth rates calculated from cell
numbers. Hence, the individual
cell volume
increased when Scenedesmuswas grown in the
biotest (static cultures) compared to the chemostat. Variability
of growth rates between
treatments and across experiments was low.
Coefficients of variance were 55%. For example, mean (&SD) growth rates of all treatments of experiment A (listed in Table 1) are
1.35kO.04 d-’ f or volume and 1.05 +0.05 d-l
for cell numbers (n = 10). We calculated a oneway ANOVA for four concentrations of Daphnia factor (0, 50, 100, and 200 liter-l) and the
controls. There was no significant difference
for volume-based growth rates (F4,10 = 2.50;
P = 0.110). The average mean growth rate was
1.363kO.016 d-l (n = 5). On the basis of cell
numbers, the ANOVA indicated a significant
difference between treatments (F4,10 = 9.48; P
= 0.002). A post-hoc test (Tukey) showed that
this difference was due to a single treatment.
However, the deviation was minimal (< lo%),
and the ANOVA was significant only because
of the small within-group variability. The mean
growth rate was 1.058+0.062 d-l (n = 5).
Daphnia
induces algal colonies
Table 2. Multiple-range
ANOVA
for mean particle
volumes (+ 1 SE, n = 3) from Exp. B. Effect of varying
numbers of fed and starved Daphnia in the incubation
water (2 ml added to 48 ml of algal culture). “Boiled”
indicates that the incubation water was heated to 100°C
before adding it to the algal culture. Overall ANOVA: F,,,6
= 39.5; P < 0.0001. Asterisks as in Table 1.
Treatment
Control
Algal homogenate
Mean particle
volume
Homogeneous
130.7(2.3)
135.3(1.9)
*
*
134.7(0.7)
177.6(7.4)
151.3(7.3)
173.7(9.4)
284.3( 18.9)
255.3(11.3)
*
Daphnia liter-’
50,
50,
100,
100,
200,
200,
starved
fed
starved
fed
fed
fed, boiled
*
*
*
*
*
*
Table 3. Multiple-range
ANOVA
for mean particle
volumes ( f 1 SE; n = 3) from Exp. C. Effect of ammonium
addition (final concn), varying incubation times of Duphniu, and addition of varying amounts of incubation water
to algal cultures. “ST” denotes starved during incubation.
Overall ANOVA: F,,, 8 = 80.9; P < 0.0001. Asterisks as
in Table 1.
Mean particle
volume
Treatment
groups
*
1547
Homogeneous
Control
158.3(4.5)
*
Ammonium-N
0.1 mg liter-’
1.0 mg liter-l
136.3(1.7)
163.0(5.0)
*
*
200 Daphnia liter-l
*
*
Although the five experiments (A-E) were
spread out over 2 months (18 March-l 3 May),
differences in the growth rates of the controls
were negligible for all of them. A one-way
ANOVA for volume-based growth rates of the
controls of four experiments (no initial value
available for Exp. D) yielded significant differences (F3,8 = 67.7; P < 0.001). Tukey’s test
showed two homogeneous groups (AB and CE).
However, the differences were small, and the
average mean growth rate (1.321kO.072 d-l;
n = 4) was similar to the values obtained in
Exp. A. Even the controls of additional experiments performed several months later (unpubl. data) did not differ from these values;
hence, the biotest proved to be reliable.
Induction strength -Absolute values of mean
particle volume can only be compared within
experiments where a single source of Daphnia
factor was used. Small differences in the size
of the daphniids, their food availability,
or
other conditions of the Daphnia cultures may
have affected concentration and activity of the
colony-inducing
factor and have caused differences between otherwise identical treatments in succeeding experiments.
The induction of colonies depends on the
concentration
of Daphnia factor (Table 1).
When the incubation water of 200 Daphnia
liter-l was serially diluted, the effect disappeared at 50 liter- I. Fifty Daphnia liter- l seems
to be the threshold concentration under our
experimental conditions, as it still produced a
significant effect in Exp. B (Table 2). The ad-
2
2
2
2
5
10
ml
ml
ml
ml
ml
ml
(12
(24
(48
(48
(48
(48
h)
h)
h)
h, ST)
h)
h)
3 16.7(6.9)
328.7( 18.8)
346.0( 10.7)
250.3(7.3)
366.3(19.3)
490.0(23.1)
groups
*
*
*
*
*
*
dition of a second portion of incubation water
after 24 h of algal growth increased the mean
particle size in the 50 liter - l treatment but not
in the undiluted samples. However, this does
not mean that the maximum effect is obtained
at 200 liter-l because the mean particle volume can be further increased if 5 or 10 ml of
incubation water (200 liter-l) are supplied instead of 2 ml (Table 3).
The colony-inducing
factor must be released
from live Daphnia because homogenate of
daphniids equivalent to a concentration of 200
liter-l had no effect (Table 1).
Actively feeding Daphnia produced more of
the colony-inducing
factor than starved ones
(Tables 2,3), although the effect was significant
only at the highest concentration (200 liter-l).
However, the colony-inducing
factor is probably not a constituent of the Scenedesmuscells
themselves, because algal homogenate was not
effective (Table 2).
Table 3 shows the effect of varying incubation times of Daphnia. The culture medium
was nearly saturated with Daphnia factor after
24 h. Incubation of the daphniids for 48 or 72
h yielded only a small (insignificant) increase
in colony formation.
Hence, we incubated
Daphnia for 24 h in all other experiments.
Nature of the colony-inducing factor - During incubation, daphniids excrete ammonium,
so we suspected that colony formation might
be induced by ammonium. At 200 Daphnia
1548
Lampert et al.
Table 4. Multiple-range
ANOVA
for mean particle
volumes (+ 1 SE; n = 3) from Exp. D. Effect of biochemical
treatments of incubation water before addition to algal
cultures. Positive control: addition of untreated incubation water. Overall ANOVA: F6,14 = 62.5; P < 0.0001.
Asterisks as in Table 1.
g
0.8
E
< 0.6
>
z
g 0.4
.t:
E
E! 0.2
a,
0.0
0
500
1000
1500
2000
Volume
Fig. 3. Test of the effect of ultrafiltration
(500 Da) on
the Daphnia factor. Particle distributions as in Fig. 1. Broken line-negative
control (no Daphnia factor); thin linepositive control (untreated Daphnia factor); thick linetreated Daphnia incubation water. Upper panel-Filtrate.
Lower panel-Supernatant.
The chemically active substance is in the filtrate (i.e. ~500 Da).
liter-l,
the incubation water contained 0.18
mg NH4-N liter-l. Thus, the addition of 2 ml
of incubation
water increased the ammonium-N concentration in the algal cultures by
- 15 pg liter-l. Ammonium concentrations of
100 hg N liter-l and 1 mg N liter-l, however,
had no effect on colony formation (Table 3).
Negative results were also obtained with urea
at concentrations of 0.8, 8.0, and 80 pg liter-l.
We attempted to destroy the Daphnia factor
relative to positive (untreated) controls. Figure
3 demonstrates the procedure with results of
the ultrafiltration
(500 Da). After ultrafiltra-
Treatment
Mean particle
volume
Negative control
Positive control
138.7(3.8)
339.7(5.4)
*
Ultrafiltration
Supernatant
Filtrate
176.7(9.1)
359.0(21 .O)
C18, run through
pH 1, 60°C
pH 12, 60°C
169.7(9.2)
363.7(18.1)
315.0(11.5)
*
*
*
Homogeneous
groups
*
*
*
tion of incubation water, the filtrate induced a
response identical to the positive control, while
the supernatant was no longer chemically active (Table 4). Hence, the inducing factor must
have a molecular mass < 500 Da.
Most of the chemical activity disappeared
when the water was passed through a Cl8 solidphase adsorption cartridge (Table 4), but it
could not be recovered by desorption of the
cartridge with methanol (Table 5).
Treatment with Pronase E did not destroy
the chemical activity of the colony-inducing
factor. Daphnia incubation water treated with
the protease induced a mean particle size (&
SD) of 264k10.6 pm3 and 3.9kO.2 cells aggregate - l; controls with protease had only
108.3k2.1 pm3 and 1.08+0.01 cells aggregate-l (n = 3).
The colony-inducing factor is heat stable and
pH insensitive. Boiling the incubation water
for a short time resulted in no significant loss
of chemical activity (Table 2). Further tests
showed that the factor is probably a nonvolatile organic substance, as it could be dried
and resuspended without losing the chemical
activity but was destroyed by incineration (Table 5).
Discussion
In the green alga Scenedesmus (Chlorococtales, Chlorophyta), cells divide inside a mother
cell and leave through an opening in the cell
wall (Van den Hoek et al. 1993). The daughter
cells may either separate to form unicells or
stay together in colonies (coenobia). Colony
Daphnia
induces algal colonies
1549
Table 5. As Table 4, but from Exp. E. Overall ANOformation is evidently a phenotypic trait conVA: J’qo = 80.2; P < 0.0001.
trolled by environmental
factors. Although
there may be differences in genotype x enviMean Particle
Homogeneous
Treatment
volume
groups
ronment interactions
(Wood and Leatham
1992), phenotypic change in colony size occurs
Negative control
135.0(2.1)
*
*
Positive control
229.0(6.1)
in several species of Scenedesmus (Trainor
*
Dried
208.3(6.2)
1993b). Hessen and van Donk (1993) were the
141.3(3.5)
*
first to show that a chemical factor released by Incinerated
C18, methanol eluate
149.7(4.8)
*
Daphnia was able to induce colonies in the
spine-armored S. subspicatus. We found an
identical response in the nonarmored S. acuadvantage from colony formation, we can astus. Hence, we hypothesize that the potential
factor evokes a
for grazer-induced colony formation is also sume that the colony-inducing
general physiological response independent of
widespread in the genus.
The direct response of Scenedesmus to the actual cell size or degree of spine formation.
If colony formation is only phenotypically
(chemical) presence of a grazer suggests an
analogy with the morphological
changes in induced when a chemical signals the presence
zooplankton in response to kairomones re- of grazers, one might expect costs associated
with colonial growth (Dodson 1989). These
leased by their predators (Larsson and Dodson
1993). These morphological changes are sup- costs do not seem to be associated with algal
posed to be protective when predator densities
growth rate (any growth rate reductions must
vary in space and time (Dodson 1989). Most
be very small), because we could not detect
grazers are size selective, so colony formation
differences between treatments and controls for
may be a grazer defense.
volume-specific
growth rates and cell multiplication rates. The same was observed by
Hessen and van Donk (1993) found, in fact,
lower grazing rates for relatively small D. mag- Hessen and van Donk (1993). However, large
na when the share of colonies in the algal food colonies must have higher sinking rates (Reynolds 1984). It is interesting that Trainor (1993a)
was high. We performed grazing experiments
with small (1.0 mm) and large (2.5 mm) D. found colony formation at cold temperatures
magna using a dual-label approach (Lampert
and unicells at warm temperatures. Due to the
higher viscosity of the water, the size effect on
and Taylor 1985) with mixtures of induced
and noninduced cells that had been altemathe sinking rate may be tolerable at low but
tively labeled with 14C or 32P. We also mea- not at high temperatures. The tradeoff between
sured filtering rates by using separate 14C-la- sinking and grazer resistance would favor a
beled algal treatments. Contrary to Hessen and direct phenotypic response to the presence of
van Donk (1993), we did not detect differences
grazers, as would be expected in a variable
environment (Schlichting 1989). In warm wain the uptake of unicells and colonies. With
our present knowledge, we can only speculate
ter, when sinking losses are important, coloon the reasons for this discrepancy. Daphnia
nies will be formed only when mortality
is a rather nonselective filter-feeder but has an through grazing is high.
upper limit of particle sizes that it can ingest
Until now, grazer-induced colony formation
(Lampert 1987). Hessen and van Donk (1993)
has been demonstrated only in the laboratory.
assumed that spined colonies of S. subspicatus To prove its adaptive value, the mechanism
must be demonstrated in the field. The miniwere larger than the maximum size that could
be ingested by a 1.75-mm daphniid. Our strain
mum concentration
of the colony-inducing
factor found to be chemically active in our
of S. acutus does not have spines, and it may
therefore be ingestible even if it forms colonies.
experiments was equivalent to a Daphnia biomass of -0.5 mg dry wt liter-l.
Such bioBecause the maximum size of ingestible parmasses are not uncommon during times of
ticles depends on the size of the daphniid (Bums
heavy grazing (Lampert 1988). The critical
1968), a reduction of feeding rates can probably be seen only in individuals that are smallconcentration in the field may even be lower
er than the ones we tested. Since our Scene- when algae have more than 48 h to react.
desmus strain does not seem to gain an Hence, it is likely that the mechanism works
1550
Lampert et al.
under natural conditions. Careful analysis of
phytoplankton and zooplankton field samples
and transplantation experiments may provide
more evidence. Trainor (1992) cited several
transplantation
experiments that resulted in
phenotypic changes in Scenedesmus. For example, unicells of S. armatus formed colonies
after 10 d of incubation in the field according
to Trainor and Roskosky (1967). At that time,
they did not suspect the presence of a chemical
signal in the field, but carefully designed experiments can now test the hypothesis, especially if the substance can be identified.
The phenotypic response of Scenedesmus is
the first experimental
proof that there is a
chemical signal from grazers that induces morphological changes in phytoplankton, although
it has been suspected (Lynch 1980). Other phytoplankton may have similar responses. Further studies will show whether the response is
unique to chlorococcal algae (or even Scenedesmus) due to their special development or
whether it is typical for other algae as well.
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Submitted: 31 January 1994
Accepted: 19 April 1994
Amended: 25 May 1994