Limnol. Oceanogr.

, 42(2), 1997, 357-364

0 1997, by the American Society of Limnology and Oceanography, Inc.

Altered cell wall morphology in nutrient-deficient phytoplankton and its

impact on grazers
E. Van Donk and M. Liirling
Department of Water Quality Management and Aquatic Ecology, Agricultural University Wageningen, POB 8080, 6700
DD Wageningen, The Netherlands

D. 0. Hessen
Department of Biology, Division Limnology, University of Oslo, POB 1027 Blindern, 0316 Oslo, Norway

G. M. Lokhorst
University of Leiden, Research Institute Rijksherbarium/Hortus Botanicus, POB 9514, 2300 RA Leiden, The Netherlands

Abstract
Grazing experiments were performed with the zooplankters Daphnia pulex and Daphnia magna feeding on
nitrogen- and phosphorus-limited cells of two green algae (Chlamydomonas reinhardtii and Selenastrum cupricor-
nutum). To analyze the role of the cell wall structure in digestibility of the algae by Daphnia, the sameexperiments
were carried out with both wild-type C. reinhardtii and a cell wall-deficient mutant.
The nonlimited algae were efficiently assimilated, whereas P- and N-limited algal cells were not. Especially
P-limited cells passedmostly intact and viable through the gut and were thus spared from heavy grazing pressure.
In life-history experiments, D. pulex grazing on nonlimited algae reached the largest body size, whereas animals
fed N- or P-limited algae exhibited reduced growth. Cells of the wall-deficient mutant of Chlamydomonas, grown
under both nutrient-limited and nonlimited conditions, were efficiently ingested and digested by Daphnia. Morpho-
logical changes in the cell wall of nutrient-limited cells most probably reduced their digestibility. This phenomenon
might be considered a defense mechanism by the algae to reduce grazing pressure when their growth rates are low.

Herbivores depend on food resources that are normally
nutritionally deficient in relation to their needs. When com-
paring plant and grazer stoichiometry, there is a common
mismatch between the somatic need for nitrogen or phos-
phorus relative to carbon in the grazer and that provided by
the plant (McNaughton 1988; Hessen 1992). In pelagic sys-
tems, the C : N or C : P ratios of phytoplankton, particularly
under nutrient limitation, often exceed those of most pelagic
grazers (Sterner and Hessen 1994). Even though assimilation
efficiency of C in algal cells can be low, there is often a
deficiency of N and P in net accumulated food compared
with the grazers’ stoichiometry (Sterner and Hessen 1994),
leading to the assumption that the growth of zooplankton
grazers is limited by minerals (Hessen 1992; Urabe and Wa-
tanabe 1992). This view is supported by the common ob-
servation that zooplankton, notably Daphnia, feeding on
P-deficient algae have reduced growth rates and depressed
fecundity (Sommer 1992; Urabe and Watanabe 1992; Sterner
et al. 1993), while P-deficient algae are sufficient for main-
tenance (Sterner and Robinson 1994). However, these stud-
ies do not constitute proof of causality, and there is no doubt
that the biochemical make-up of the food (like the content
of unsaturated fatty acids) can also play a major role (Ahl-
gren et al. 1990; Miiller-Navarra 1995).
Acknowledgments
We thank the staff at the Norwegian Institute for Water Research
for the use of algal cultures and laboratory facilities, W. Star for
making the TEM photos, W. Lukassen for her assistance during the
life-history experiments, and R. D. Gulati and B. A. Faafeng for
their constructive comments on the manuscript.
357
An additional mechanism that may contribute to the re-
duced success of Daphnia feeding on P-limited algae was
proposed by Van Donk and Hessen (1993). Following the
observation that both Daphnia pulex and Daphnia magna
exerted lower grazing pressure on P-starved green algae (Se-
lenastrum capricornutum and Scenedesmus subspicatus) rel-
ative to nutrient-saturated algae, Van Donk and Hessen
(1993) found that P-starved cells passed largely intact
through the daphnid gut. Typically, the P-starved algae in-
creased their cell size, probably owing to arrested cell di-
vision and accumulation of intracellular glycogen com-
pounds. In a follow-up study with D. magna feeding on S.
capricornutum, Van Donk and Hessen (1995) confirmed this
mechanism and demonstrated the same effect in UV-B-ir-
radiated algae.
Similar observations have been interpreted as decreased
ingestion rates (Sterner and Smith 1993; Sterner et al. 1993).
However, part of these reductions may have been the result
of varying ability to assimilate nutrient-limited cells, because
they determined clearance rates in long-term experiments
(-8 h). The observation of a major whole cell gut passage
led Van Donk and Hessen (1993) to conclude that the effect
could be largely accredited to increased resistance of the
algae to the grazers’ digestive enzymes. Most phytoplankton
species accumulate excess C as starch granula, whereas in
some phytoplankton and bacteria species, mineral nutrient
deficiency leads to accumulation of excess C as extracellular
polysaccharides (Myklestad 1977; Sondergaard and Schienip
1982), which may also block digestive enzymes (Malej and
Harris 1993).
358 Van Donk et al.
Planktonic algae are able to withstand grazing pressure
from zooplankton in various ways. The most obvious way
is through changes in morphological features such as size or
cell wall shape. There is a tradeoff between the metabolic
costs associated with morphological changes produced for
grazing protection and growth rate. With mineral nutrient
supply in excess, fast growth rates may to some extent com-
pensate for grazing losses. In nutrient-deficient systems,
however, growth rate slows and some morphological means
of grazer protection would be more beneficial, even at the
expense of growth rate. For some algal species, increased
cell wall thickness under unfavorable conditions may be
seen as a preparation for encystment; one can hardly claim
that thickening of the cell wall is a primary strategy to de-
press growth of grazers, yet it may have exactly that effect.
Hence, for a number of reasons, nutrient-deficient phyto-
plankton constitute poor food for zooplankton, and such a
nutrient shortage at the base of the food web will have an
impact on the entire food web and adversely affect second-
ary production at all levels.
In this study, the use of a mutant clone of the green algae
Chlamydomonas reinhardtii, which lacks a cell wall, al-
lowed a direct test of the role of algal cell wall properties
on Daphnia grazing and growth. We found that morpholog-
ical changes in the cell wall of nutrient-limited algal cells
reduced their digestibility and that animals feeding on these
algae exhibited reduced growth.
Methods
Planktonic organisms-The green algae S. capricornutum
NIVA CHL 1, C. reinhardtii NIVA CHL 13 (wild-type
strain), and C. reinhardtii NIVA CHL 75 (cell wall-deficient
mutant) were used in the experiments. These algal species
are kept in cultures at the Norwegian Institute for Water
Research. The zooplankters D. pulex and D. magna were
used for the grazing experiments.
Pretreatment oj’ phytoplankton-Inoculum phytoplankton
cultures were incubated in the inorganic nutrient medium 28
20% at 20°C; for the Chlamydomonas mutant, 28 medium
was modified according to Skulberg and Skulberg (1990).
Illumination was provided by cool-white fluorescent tubes at
70 pmol quanta m-* s-l at 14: 10 L/D cycle. To obtain P-
and N-limited algal cells, we inoculated exponentially grow-
ing cells into flasks containing a P-free medium and N-free
medium, respectively. The cells entered stationary state after
5 d (P limitation) and 3 d (N limitation). At the start of each
grazing experiment the C, N, and P contents of the algae
were analyzed. C and N contents were measured on a Carlo-
Erba CHN 1106 elemental analyzer and P content on the
total samples after peroxo-disulfate digestion. Cell volumes
of the algae were determined in a Coulter Multisizer II (100
,um capillary).
Transmission electron microscopy-To study changes in
the morphology and structure of the algae, we made trans-
mission electron microscopy (TEM) photos of the algal cells
cultured under different nutrient conditions. The algae were
initially concentrated by gentle centrifugation to obtain pel-
lets that were fixed in 2% glutaraldehyde in 0.1 M sodium
cacodylate buffer (pH 7.6) for 1.5 h at room temperature.
After resuspension and rinsing in buffer, cells were again
concentrated. by gentle centrifugation, collected on cellulose
nitrate filters (Schleicher and Schtill; pore size 0.8 pm), and
covered by #agar.The filters with the attached layer of cells
in agar were cut into small pieces, postfixed in 1% 0~0, in
0.1 M sodium cacodylate for 1 h at room temperature, rinsed
in distilled water, dehydrated with en bloc staining in 1%
uranyl acetate in an ethanol series, and finally embedded in
Epon. Seria.1 sections were cut with a diamond knife on a
LKB ultratome and mounted on pioloform-coated slot grids.
Specimens were poststained with uranyl acetate and lead ci-
trate and examined with a JEOL lo-10 electron microscope.
Grazing experiments-The grazing experiments were car-
ried out in OS-liter Erlenmeyer flasks, containing 200 ml of
medium. The initial algal cell concentration was -2X 10”
cells ml-l for Selenastrum and lo5 cells ml-’ for Chlamy-
domonas. In our first experiment we studied D. pulex feeding
on S. capric,ornutum and C. reinhardtii (with cell wall), cul-
tured under non-, P-, and N-limited conditions. In our second
experiment we fed D. magna both algae. Our third experi-
ment was performed with D. magna grazing on non-, P-,
and N-limited mutant C. reinhardtii (lacking cell wall). At
the start of Exp. 1, 12 similar-sized D. p&x (length 2.0-2.5
mm, weight 34-67 pg C) were added to 200 ml of each
algal monoculture. For the experiments with D. magna (Exp.
2 and 3), 1:hree individuals (mean length 3.5 mm, mean
weight 160 pg C) were incubated in the 200-ml algal sus-
pension. Before incubation, the animals were washed to re-
move surface contamination. The grazing experiments, last-
ing 2-3 d, were carried out in duplicate. Control flasks were
without animals. Flasks were shaken manually four times a
day. Samples for algal counts in the experimental flasks were
taken at t == 0, 19, 43, and 67 h in Exp. 1 and at t = 0, 19,
24, 43, 48, and 67 h in Exp. 2 and 3 (Daphnia introduced
at t = 0). Samples were preserved with Lugol’s solution.
Algae were counted microscopically and least-squares re-
gressions of ln(cells ml-‘) vs. time (h) were calculated. Be-
cause measurements for replicate flasks were close compared
to other sources of variation, replicates were pooled. The
slopes of the regressions for the controls (algal growth rates)
plus the disappearance rates in the grazing experiments gave
the apparent clearance rates. These apparent clearance rates
are not typical clearance rates because a great proportion of
algae in th,e nutrient-deficient treatments were not digested
and were hence recounted. Fecal analyses of the daphnids
accompanied each experiment. For this purpose animals
were placed individually in a drop of water on slides under
coverslips. After the animals had defecated, rectal contents
were dispersed and examined microscopically (Porter 1975).
Viability experiments- To study viability of algal cells in
the feces o-FDaphnia, we incubated D. magna in suspensions
of P-limited and nonlimited cells of Chlamydomonas. After
10 min the daphnids were successively placed in five Petri
dishes with Z8 medium without algae to remove contami-
nation by surface-attached algae. This step took less than 3
min. The daphnids were then transfered to flasks with 25 ml
 Altered cell wall morphology 359

of complete 28 medium and removed again after 15 min. Table 1. Apparent clearance rates of algae (ml ind.-’ h-l) (SE
These flasks with egested material of daphnids were then in parentheses) as measured in three experiments with zooplankters
incubated for 5 d at 20°C and at an illumination of 70 pm01 Daphnia pulex (60 ind. liter-’ in Exp. 1) and Daphnia magna (15
quanta mm2 s-l on a 14: 10 L/D cycle. At days 0, 1, 2, 3, ind. liter-’ in Exp. 2 and 3). In Exp. 1 and 2 grazing was on non-,
N-, and P-limited Chlamydomonas reinhardtii (C.r.) and Selenas-
and 5 the number of cells were counted with a Coulter Coun-
trum capricornutum (SC.). In Exp. 3 grazing was on a cell wall-
ter. The experiment was performed in triplicate. deficient mutant of C. reinhardtii (m.C.r.).
Feeding rates- To study the effect of P- and N-limited C:N:P Apparent
Chlamydomonas on feeding rates of Daphnia, we conducted in algae clearance rate
experiments with D. pulex grazing on a mixture of algae and Treatment (atomic ratios) (ml ind.-’ h-l)
polystyrene beads. The beads were used to analyze whether Exp. 1
the reduction in clearance rates, found for nutrient-limited
C. r. (nonlim.) 214:31: 1 0.69(+0.15)
algae, could be explained by an increased resistance of these C.r. (P lim.) 845:99: 1 0.02( 20.09)
algae to assimilation or by a reduced feeding rate of the C.r. (N lim.) 665: 1 0.23( 20.02)
animals. At least 1 h prior to the grazing experiments, a SC. (nonlim.) 69:lO:l 1.50(+0.11)
cohort of D. pulex (-2.7 mm) was transferred into mem- S.C. (P lim.) 408:33: 1 0.16(?0.06)
brane-filtered Lake Maarsseveen water to empty their guts. S.C. (N lim.) 182:14:1 1.05(+0.12)
Thereafter, feeding rates of these animals were measured on Exp. 2
a mixture of C. reinhardtii varying in quality and polysty- C. r. (nonlim.) 223:25: 1 2.94(+0.17)
rene beads (particle diameter 2 1.6 pm). The algal food types Cr. (P lim.) 611:85:1 0.33(?0.12)
(C. reinhardtii) were nonlimited, N-limited, and P-limited Cr. (N lim.) 73:4: 1 0.83(&0.22)
algae. Five adult animals (-2.7 mm) were placed in 25 ml S.c. (nonlim.) 67:9: 1 5.89(&0.24)
of food suspension, a mixture of similar volumes of algae S.C. (P lim.) 714:72: 1 0.61(10.22)
(1.8X106 pm3 ml-l) and polystyrene beads (1,100 beads S.C. (N lim.) 55:2: 1 2.94(50.22)
ml-‘) in 0.45-pm membrane-filtered lake water. Statistical Exp. ‘3
optimization of the experiment revealed that at least 26 an- m.C.r. (nonlim.) 15:2: 1 7.25( 20.58)
imals per food type had to be used. Hence, for each food m.C.r. (P lim.) 49:4: 1 6.94( “0.47)
type, 6 replicates with 5 or 6 animals per vessel were ap- m.C.r. (N lim.) 36:2: 1 6.38(?0.33)
plied. After a 5-min grazing period in the dark at 2O”C, the
animals were rapidly transferred into and rinsed in CO,-sat-
urated water. Narcotized animals were placed on a glass slide cell, P- and N-limited algae were added at similar volumes
and dissolved in concentrated sulfuric acid (according to as used in the series with nonlimited Chlamydomonas. The
Bern 1990) and polystyrene beads were counted with the test tubes with the experimental animals were incubated at
light microscope (100X magnification). The mean numbers 20°C in the dark to prevent algal growth. The animals were
of ingested beads per food type were compared statistically transferred daily into clean tubes containing fresh medium
by one-way ANOVA. Clearance rates (CR) (ml ind.-’ h-l) with similar pH (8.25rt0.12) and conductivity (450524 PLS
were calculated from the number of ingested beads in rela- cm-l). Body length was measured and the animals were ex-
tion to suspended beads: amined daily for molting. Time needed to reach maturity,
Beads ind.-’ number of newborns, and length of newborns were recorded.
CR = x 6o Newborns were removed from the tubes. Growth and repro-
Beads ml-l 5’ duction were measured until the animals reached the fourth
Life history experiments-These experiments were carried adult instar and consequently had released their third clutch,
out to determine population growth rate of D. pulex when because the population growth rate is mainly determined by
feeding on C. reinhardtii (with cell wall), cultured under the first three clutches (Vanni and Lampert 1992). The in-
non-, P-, and N-limited conditions. A life-table test with both trinsic rate of population increase (r) was estimated using
Chlamydomonas clones (with cell wall and mutant cells), the Euler equation:
growing in log-phase and under nutrient limitation, would
also have allowed us to distinguish between effects of re- 1 = 2 exp(-rx)l,m,,
x=0
duced assimilation and a biochemical/mineral mismatch.
However, we were unable to maintain the nutrient-limited, where r is the rate of population increase (d-l), x is age class
cell-wall-deficient clone stable in culture for a sufficient time (0 . . . N), 1, is probability of surviving to age x, and m, is
to conduct life-history experiments. fecundity at age x.
The experimental animals were produced by a cohort of Owing to the impossibility to compute standard errors of
Daphnia females cultured at a food level of 2 mg liter-l C the population parameter r directly, a Jackknife method was
nonlimited Chlamydomonas. Newborns from the third clutch used to calculate standard errors (Meyer et al. 1986).
were collected within 20 h of birth and joined in a 500-ml
beaker. For each series, 20 neonates were randomly selected Results
from this beaker and placed individually in loo-ml test tubes
containing 60-ml of non-, N-, or P-limited C. reinhardtii. Grazing experiments- The apparent clearance rates for D.
Because of dissimilar morphology and varying C content per pulex and D. magna feeding on P-limited and N-limited algal
 Altered cell wall morphology
cells in Exp. 1 and 2 were greatly reduced, compared with
the rates on nonlimited algae (Table 1). There was a reduc-
tion of -9O-95% of the apparent clearance rate for P-limited
and -3O-70% for N-limited cells of Selenastrum and
Chlamydomonas. In Exp. 3, D. magna feeding on P-limited
and N-limited cells of Chlamydomonas mutant showed no
significant reduction in apparent clearance rates compared
with the nonlimited cells (Table 1). The clearance rates were
twice as high for the mutant as for the nonlimited Chlamy-
domonas with cell wall.
Transmission electron microscopy-The TEM photo-
graphs show that irrespective of the nutrient composition of
the culture medium, the cell wall of the Chlamydomonas
cells is composed of an inner and an outer layer (Fig. 1.5,
1.6). The inner layer (Wl) is amorphous and electron-lucent.
The outer layer is connected firmly to the outer surface of
Wl and is composed of several sublayers including the elec-
tron-dense W2 and W6 (Fig. 1.5, 1.6) (see also Roberts et
al. 1985). P-and N-limited cells have a thicker cell wall com-
pared to nonlimited cells, which is apparently caused by
swelling of Wl (Fig. 1.3, 1.4, and 1.6). The TEM photo-
graphs of the nonlimited cells are self-explanatory (Fig. 1.1,
1.2). Additionally, TEM observations show that the chloro-
plast of the nutrient-limited cells is strongly obscured by
heavy accumulation of storage products. In N-limited cells
the pyrenoid is often difficult to detect (Fig. 1.4). Apparently,
these changes in structure would not serve as a morpholog-
ical constraint for ingestion by Daphnia, but may reduce the
assimilation efficiency.
Feces analyses and viability experiments-Feces analyses
revealed that P-limited and to a lesser extent N-limited algal
cells of Selenastrum and Chlamydomonas (both with cell
wall) passed mostly undamaged through the guts of the
daphnids, probably owing to reduced digestibility. Under the
light microscope the presence of large starch grains was seen
in nutrient-limited algae and an increase in cell volume (Ta-
ble 2). The number of Chlamydomonas cells still viable in
the feces of Daphnia after gut passage is shown in Fig. 2.
The feces of daphnids fed P-limited cells contained high
numbers of intact algal cells, but for animals fed nonlimited
cells viable algal cells were very scarce and observed only
after 2 d of incubation. The growth rate of defecated P-lim-
ited cells was not significantly different from the rate of
nonlimited cells.
Feeding rates-In the presence of different food types
(non-, N-, or P-limited algae) the uptake of polystyrene
beads did not differ significantly (F2,9, = 0.78; P = 0.465),
and hence clearance rates based on bead ingestion were sim-
ilar (Table 3). Also, the number of beads in the daphnid guts
did not differ statistically (Table 3). Hence, Daphnia did not
reduce its feeding rate in response to nutrient-deficient cells.
t
Fig. 1. Longitudinal sections of cells of Chlamydomonas reinhardtii (TEM photos). 1. Nutrient-saturated cell (X 8,800). 2. Wall-deficient
mutant (X8,480). 3. P-limited cell (X7,000). 4. N-limited cell (X8,500). 5. Detail of wall of nonlimited cells (X85,800). 6. As panel 5,
but of P-limited cell. Abbreviations used: C, chloroplast; FA, flagellar apparatus; N, nucleus; P pyrenoid; S, starch; ST, stigma; W, sublayer
of wall (numbered according to the convention of Roberts et al. 1985).
361
Table 2. Cell volume (pm’) (SE in parentheses) of algal cells
of Selenastrum carpicornutum, Chlamydomonas reinhardtii, and
Chlamydomonas reinhardtii (mutant) cultured under different con-
ditions.
Non- N P
limited limited limited
S. capricornutum 31.0(+2.5) 39.0(&3.4) 60.0(+ 1.8)
C. reinhardtii 97.3(&8.8) 204.3(&28.2) 153.4(+22.3)
C. reinhardtii (mutant) 71.6(+3.5) 76.4(?3.8) 77.8( k2.5)
The reduction in apparent clearance rate can be largely at-
tributed to a decreased assimilation of the nutrient-deficient
algal cells.
Life-history experiments -D. pulex grazing on nonlimited
Chlamydomonas reached the largest size, while in those fed
N- or P-limited algae the increase in size was significantly
less (Fig. 3). The intrinsic rate of population increase (r) was
significantly different for the three treatments (F2,54= 143.1;
P < 0.001); th e h’ig h est value recorded was for D. pulex
grown on nonlimited algae and the lowest for those fed with
P-limited algae (Table 4). The duration of the juvenile period
was similar for Daphnia fed non- or N-limited algae
(5.850.6 and 5.810.5 d, respectively), but was higher for
those fed P-limited algae (6.62 1.3 d). Food quality signifi-
cantly affected the size at maturity (F2,50 = 109.6; P <
0.001) (Table 4) and the number of total newborns produced
by the three observed adult instars (Fig. 4).
Discussion
The data for D. magna grazing on two clones of C. rein-
hardtii indicate that the cell wall of the alga, especially under
nutrient-limited conditions, forms a barrier for digestion by
zooplankton. The cells became less digestible, probably due
to morphological changes. The increase in cell volume and
wall thickness of nutrient-limited algae may reflect a storage
of proteins, carbohydrates, and lipids probably owing to de-
layed cell division. Increased cell volumes and a granular
appearance have also been noticed in other algae cultured
under nutrient limitation (Mitchell et al. 1992; Sterner et al.
1993; Van Donk and Hessen 1993). Myklestad (1977) and
Sondergaard and Schierup (1982) observed the release of
photosynthetic products by algae growing under nutrient
stress. Such extracellular mucous layers may also block di-
gestive enzymes (e.g. Malej and Harris 1993). The two lay-
ers in the cell wall of Chlamydomonas are both composed
entirely of glycoproteins (Roberts et al. 1985). During nu-
trient limitation, the inner wall layer, which becomes thicker
(see Fig 1.6: Wl), is rich in hydroxyproline and contains a
high percentage of carbohydrates. Furthermore, this layer is
362 Van Donk et al.

13 ,

l Nonlimited
o N limited

: m 0 2 4 6 8 10 12 14 16

01 I I I I I 1
0 1 2 3 4 5
Time (d)
Fig. 2. Cell density of Chlamydomonas cells with cell wall
(P-limited and nonlimited) still viable in the feces of Daphnia. Lin-
ear regression lines are presented-for nonlimited cells, y = 0.90x
+ 5.12 (R2 = 0.77); for P-limited cells, y = 1.03x + 0.65 (R2 =
0.69).
amorphous and very insoluble (Home et al. 1971). The mu-
tant of Chlamydomonas does not form a normal cell wall,
but secretes its cell wall glycoproteins into the culture me-
dium (Lang and Chrispeels 1976).
The results of the grazing and viability experiments as
well as microscopic analysis of the algal cell wall and feces
of daphnids indicate that the ingestion rates of nutrient-lim-
ited algae and nonlimited algae are similar but that nutrient-
limited algae are poorly digested. In the literature the ability
of various species of zooplankton to sense nutrient-limited
algal cells has been discussed as a mechanism that allows
them to discriminate against potentially low-quality food.
Several species of filter-feeding copepods are known to dis-
criminate against N-deficient cells (Cowles et al. 1988; But-
ler et al. 1989; Kiorboe 1989). However, daphnids lack such
active selection in mixtures (DeMott 1986), but may change
the rate of food collection (Butler et al. 1989) and apparently
also the rate of ingestion (Sterner and Smith 1993). Sterner
and Smith (1993) reported reduced clearance and feeding
rates, irrespective of food concentrations, when fed Daphnia
on nutrient-limited Scenedesmus, especially P-deficient cells.
However, some of these reductions in feeding by Daphnia,
Table 3. Ingested beads (+SE) and clearance rates based on
beads (ml ind.-’ h-l; +SE) of Daphnia pulex fed on a mixture of
Chlamydomonas reinhardtii varying in quality and polystyrene
beads (N = number of daphnids).
Ingested Clearance
Food type beads rate N Food type
Nonlimited 962 18 1.04?0.02 30
N limited 7629 0.83?0.10 32
P limited 9529 1.03?0.10 32
Time (d)
Fig. 3. Increase in length over 14 d of Daphnia pulex feeding
on non-, P-, and N-limited Chlamydomonas cells.
6
as measured by Sterner and Smith (1993), may have been
the result of varying ability to assimilate nutrient-limited
cells, because they determined feeding rates (in fact assim-
ilation rates) in long-term experiments (-8 h) by monitoring
the decline of food-cell densities.
We found that Daphnia grazing on P-limited algae exhib-
ited reduced rates of growth to maturity and reduced fecun-
dity. Mitchell et al. (1992) also found that Daphnia survived
better, grew faster, and had larger broods when fed on non-
limited Chlamydomonas than on N- or P-limited cells. They
suggested that the effect might be related to the nutritional
inadequacy of the algae, but they were not able to find a
direct relationship to the nutrient content of the cells. Other
studies have also reported decreased growth and reproduc-
tion in Daphnia if fed P- or N-limited algae even though
food was abundant (Hessen 1990; Watanabe 1990; Groeger
et al. 199 1; Sommer 1992). The observed differences in life-
history parameters of Daphnia grazing on nonlimited and
nutrient-limited algae are probably due to both a reduced
food availability, because of decreased assimilation of nu-
trient-limited algae, and a reduced nutritional content of the
algae. Th.s finding is strikingly analogous to the well-known
reduced palatability of older leaves in many terrestrial plants
due to their poor nutrient quality (Mattson 1980).
If these observations also apply to natural plankton pop-
ulations, they might constitute an important feedback mech-
anism contributing to the stability of open-water ecosystems.
Thus at tj.mes when phytoplankton growth rates are reduced
Table 4. Rate of population increase (r, d-l ? 1 SD), size at
maturity, and body length of the juveniles (mm, -I-1 SD) of Daphnia
pulex gro’nn on Chlamydomonas reinhardtii of different quality.
Asterisks not sharing the same vertical column indicate homoge-
neous groups that are significantly different at a 95% level (Tukey
test).
r (d-l) Adults (mm) Juveniles (mm)
Nonlimited 0.50+0.02* 2.5250.07 (n = 20)” 0.76kO.03
N limited 0.36t0.08 * 2.2750.09 (n = 18) * 0.75?0.04
P limited 0.21kO.05 * 2.08+0.11 (n = 17) * 0.72-LO.04
 Altered cell vu11morphology
0 N limited
1 2 3 rates: Jackknife vs. bootstrap techniques. Ecology 67: 1156
Adult instar
Fig. 4. Number of juveniles during the three observed adult
instars being produced by Daphnia pulex feeding on non-, P-, and
N-limited Chlamydomonas cells.
by nutrient deficiency, grazing pressure on the phytoplankton
would also be reduced by the depression of assimilation rates
and by the reduced rates of growth to maturity, and reduced
fecundity of the zooplankton. Moreover, viable gut passage
may be beneficial for the nutrient-limited algae, allowing
them to take up nutrients from the Daphnia gut when pools
of dissolved nutrients are depleted (Porter 1973). Finally,
these algae may become less limited and digested by the
daphnids after repeated egestions and ingestion, i.e. after
several gut passages. Apparently, nutrient limitation of the
phytoplankton can alter trophic interactions, reducing trans-
fer of energy to herbivorous zooplankton.
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