Journal of Plankton Research Vol.18 no.5 pp.757-765,1996
Combined effects of food shortage and oxygen deficiency on life
history characteristics and filter screens of Daphnia
Takayuki Hanazato1
Regional Environment Division, National Institute for Environmental Studies,
Onogawa, Tsukuba, Ibaraki 305, Japan
'Present address: Suwa Hydrobiological Station, Shinshu University, 5-2-4
Kogandori, Suwa, Nagano 392, Japan
Abstract. Daphnia magna were reared in environments with different concentrations of food and dissolved oxygen, and their life history characteristics andfilter-screenareas were analyzed. Both low food
concentration and low oxygen concentration degraded Daphnia fitness (as measured by individual
growth and reproduction), and synergism was detected statistically for the effects of the two factors
together. Low food concentration induced the development of large filter screens in Daphnia, but this
development was suppressed by low oxygen concentration, suggesting that low oxygen concentration
reduces the filtering rate of Daphnia in a food-limited environment. Therefore, energy uptake by
Daphnia seems to be reduced by food shortages and oxygen deficiencies jointly, and this reduction in
energy uptake may be a possible mechanism causing the synergistic effects of the two environmental
stresses on the life history of Daphnia.
Introduction
There are numerous environmental factors affecting the population dynamics of
the cladoceran Daphnia in nature (Peters and DeBernardi, 1987; Dodson and
Frey, 1989). Daphnia may experience some of these factors simultaneously, and
their effects are likely to be synergistic (Hanazato and Dodson, 1992,1995). Thus,
analysis of the combined effects of environmental factors on Daphnia population
dynamics is necessary to understand this organism's ecology.
Daphnia often perform diel vertical migration to avoid fish predation (Lampert,
1993a). Migrating Daphnia remain in the hypolimnion during the day, where concentrations of food and oxygen often become low (Landon and Stasiak, 1983;
Hanazato et ai, 1989). Both of these conditions degrade Daphnia fitness (Threlkeld, 1987; Nebeker et al., 1992; Hanazato and Dodson, 1995). Thus, vertically
migrating Daphnia may often experience these two detrimental environmental
factors—low concentrations of food and oxygen—together.
Low food concentrations affect the morphology of filter screens in Daphnia,
which increase in area in low-food environments (Koza and Korinek, 1985; Lampert, 1994). This change increases the efficiency of food collection, resulting in
increased filtering rates (Lampert, 1994). Daphnia may allocate more energy to
developing large filter screens in such environments than in environments with
abundant food because raising filter efficiency has a higher priority in the energy
allocation when food is limiting.
It is assumed that the energy allocation is also altered when Daphnia are
exposed to other environmental stresses such as low oxygen concentration. Since
low oxygen induces hemoglobin synthesis in Daphnia (Landon and Stasiak, 1983;
© Oxford University Press
757
T.Hanazato
Engle, 1985), which requires energy, it may change the energy allocation, thus
affecting the morphology (area) of filter screens.
I hypothesize that low food and low oxygen concentrations have combined
effects on Daphniafilterscreens, and thus on Daphnia life history characteristics.
The purpose of the present study is to analyze them.
Method
The animal studied was a clone of Daphnia magna, which had been cultured for
several years in my laboratory as a bioassay organism for toxicity tests. The stock
culture had been maintained at 23°C in aged tapwater and fed the green alga
Chlorella, which was harvested in log-phase growth.
The animals were exposed to either of two dissolved oxygen concentrations
(high or low), each with three food concentrations (high, medium or low) at 23°C in
the experiments. The high-O2 culture water (~8.5 mg O21"') was aged and wellaerated tapwater, whereas the Iow-O2 culture water (~2 mg O2 H) was prepared
by bubbling the aged tapwater with compressed N2. Each culture contained one of
three different concentrations of Chlorella (high: 1 x 106 cells ml"1 or 2 (xg dry wt
ml"1; medium: 3 x 103 cells ml"1 or 0.6 u.g dry wt ml"1; low: 1 x 105 cells ml"1 or 0.2 u.g
dry wt ml"1).
Mother individuals of D.magna were reared with Chlorella at 1 x 106 cells ml"1 at
either the high or low O2 concentrations for >2 weeks. The water in these cultures
was renewed every day. Neonates (<16 h old) of nearly the same sizes (0.730.80 mm) from the first brood of the D.magna mothers were collected and each
was placed into a glass bottle containing 125 ml of culture water with high or low
oxygen concentration (at the same oxygen condition used for the egg hatching)
with one of the three food levels. Eight individuals were prepared for each treatment. However, one individual showed extraordinarily poor growth in each of two
treatments. To avoid any effect of these atypical data on the analysis, the data for
the individual which showed the poorest growth in each of all the treatments were
discarded. Thus, there were seven replicates for each treatment. The bottles were
kept open in the treatments with high O2 and werefirmlystoppered in the treatments with low O2. All bottles were kept in an incubator at 23CC under dark conditions to prevent photosynthesis by the Chlorella.
Daphnia produce their smallest neonates in the first brood (Ebert, 1991; Lampert, 1993b), and small-bodied neonates are more sensitive to starvation and low
oxygen concentrations than are large-bodied neonates (Gliwicz and Guisande,
1992; Hanazato and Dodson, 1995). In the present study, neonates from the first
brood were used to more sensitively detect the effects of these two factors on
Daphnia.
The culture water was changed every day, at which time the body lengths of the
animals (from the top of the head to the posterior of the carapace) were measured
to the nearest 0.034 mm under a binocular dissecting microscope. When the test
animals produced their first broods, the number of eggs in the brood chamber was
determined and the individual culture was terminated. The growth rate during
juvenile stages (GR) was calculated as:
758
Effects of food shortage and O, deficiency on Daphnia
i
HfHoi
MfHox
.-
_A.
-
UHoi
a
i~2
HfLoi
J=
MfLoi
LfLax
>~>
S1
m
0
4
Instar
Fig. 1. Instar-specific growth of D.magna exposed to three food concentrations (high, medium and low)
and two oxygen concentrations (high and low). HfHox = high food-high oxygen: MfHox = medium
food-high oxygen; LfHox = low food-high oxygen: HfLox = high food-low oxygen; MfLox = medium
food-low oxygen; LfLox = low food-low oxygen.
GR = \n(MS/IB)/MT
where MS is mature size (mm), which is the size of the instar at which the animal
produced its first brood, IB is initial body length (mm), which is the size of the
neonate (first instar), and A/7"is maturation time, which is the time in days taken to
grow from birth to maturity.
To prepare samples for filter-screen measurements, D.magna were cultured at
23°C at <20 individuals 1"' in 11 glass bottles containing water with one of four
different culture conditions (high O2 + high food level; high O2 + low food level;
low O2 + high food level; low O2 + low food level). The culture water was changed
every day and the culture was continued for 2-A weeks. Individual daphnids were
harvested from each bottle at appropriate sizes and preserved in sucrose-formalin
(Haney and Hall, 1973).
The third thoracic legs were dissected off under a binocular dissecting microscope and the combs of these legs were spread out on a glass slide. The combs were
photographed and the outlines of thefilterscreens were plotted on paper. The area
of thefilterscreens was determined with a planimeter (LI-COR Model 3100). One
individual had two third leg combs, whose areas were averaged.
Results
Life history characteristics
Under the high-oxygen condition with high food concentration, the animals grew
from 0.77 mm in the first instar to 2.78 mm in the sixth instar in 5 days. This rapid
growth rate under high oxygen levels decreased with decreasing food levels (Figure 1; Table I). All the individuals matured at instar 6 (Table I), but the size at
759
T.Hanazato
Table I. Mean and SEM (in parentheses) of parameters examined for each treatment and results of
Bonferroni's multiple comparison tests. In the multiple comparison for MAT. the value of 5.1 was
assigned to one individual in the treatment HfHox to calculate a variance, because all the individuals
examined had the same value. 5. For the same reason, the value of 6.1 was assigned to one individual in
each of the treatments HfHox. MfHox and LfHox in the analysis for MAI
BL6
MAS
GR
MAT
MAI
EGG1
HfHox
MfHox
LfHox
HfLox
MfLox
LfLox
2.778(0.0312)
2.778(0.0312)
0.257(0.0026)
5.0(0.00)
6.0(0.00)
17.fr
(1.31)
2.623(0.0359)
2.623"
(0.0359)
0.211"
(0.0076)
5.9(0.14)
6.0"
(0.00)
11.4"
(1.17)
2.237"
(0.0116)
2.237'
(0.0116)
0.164'
(0.0060)
6.6"
(0.20)
6.0"
(0.00)
4.91
(0.26)
1.814'
(0.0523)
2.272'
(0.0283)
0.099"
(0.0078)
11.3"
(0.80)
8.5"
(0.43)
4.0*
(0.45)
1.86O
(0.0733)
2.336*
(0.0321)
0.093d
(0.0076)
12.7*
(1.41)
8.7"
(0.61)
1.678<
(0.0312)
2.341'
(0.0240)
0.066'
(0.0013)
16.9<
(0.40)
9.7"
(0.18)
1.9*
(0.34)
(0.26)
HfHox = high food and high oxygen concentrations; MfHox = medium food and high oxygen concentrations; LfHox = low food and high oxygen concentrations; HfLox = high food and low oxygen concentrations; MfLox = medium food and low oxygen concentrations; LfLox = low food and low oxygen
concentrations.
BL6 = body length (mm) at the sixth instar; MAS = mature size (mm) at which the first clutch was
produced; GR = juvenile growth rate (body length); MAT = time (days) to grow from birth to
maturity; MAI = matured instar at which the first clutch was produced; EGG1 = number of eggs produced in the first clutch.
Means denoted by the same letter are not significantly different from each other (P > 0.05) for the same
parameters.
maturity decreased as food concentration diminished. These differences were statistically significant (Table I).
Low oxygen concentration suppressed the animals' growth. The body lengths of
the sixth-instar individuals under low oxygen were significantly smaller than those
under high oxygen (Table I). The low oxygen delayed maturation time and
increased matured instar (at which the individuals produced their first clutches)
(Table I). This agrees with Ebert's (1992) finding that D.magna individuals with
low growth rates take more instars to mature than those with high growth rates.
Reduced food level decreased the growth rate of juveniles under both high- and
low-oxygen conditions (Table I). It lengthened the maturation time under low oxygen, but not under high oxygen. The delayed maturation time resulted from elongation of the inter-molting period of juveniles and the increased instar number of
juvenile stages. This was inferred from the following results. When the food level
was reduced from high to low under low-oxygen condition, the matured instar was
delayed by 1.2 instar number, although the delay was statistically insignificant,
while the maturation time increased by >5 days (Table I).
Maturation sizes were similar (~2.3 mm) in the four treatments: low food-high
oxygen (LfHox), high food-low oxygen (HfLox), medium food-low oxygen
(MfLox) and low food-low oxygen (LfLox) (Table I). This size (2.3 mm) may be
the minimum size for D.magna to mature. The similar minimum mature size (= the
760
Effects of food shortage and O, deficiency on Daphnia
Table II. Two-way ANOVAS with replication testing the effects of food concentration and oxygen
concentration on the six parameters. In the analysis of MAT, the value of 5.1 was assigned to one
individual in the treatment HfHox to calculate a variance, because all the individuals examined had the
same value. 5 (see Table I). For the same reason, the value of 6.1 was assigned to one individual in each
of the treatments HfHox, MfHox and LfHox for the analysis of MAI (see Table 1). Abbreviations are as
in Table I
Factor
BL6
Food
Oxygen
Interaction
MAS
Food
Oxygen
Interaction
GR
Food
Oxygen
Interaction
MAT
Food
Oxygen
Interaction
MAI
Food
Oxygen
Interaction
EGG1
Food
Oxygen
Interaction
SS
d.f.
MS
F ratio
P
0.926
6.093
0.288
2
1
2
0.463
6.093
0.144
34.560
454.637
10.740
<0.001
<0.001
<0.001
0.483
0.529
0.624
2
1
2
0.242
0.529
0.312
43.974
96.212
56.798
<0.001
<0.001
<0.001
0.032
0.161
0.002
2
1
2
0.016
0.161
0.001
65.428
659.185
4.211
<0.001
<0.001
0.023
99.889
645.116
18.747
2
1
2
49.945
645.116
9.374
15.580
201.241
2.924
<0.001
<0.001
0.067
3.570
91.319
2.073
2
1
2
1.785
91.319
1.037
2.791
142.795
1.621
0.075
<0.001
0.212
426.107
728.969
154.729
2
1
2
213.054
728.969
77.364
51.074
174.753
18.546
<0.001
<0.001
<0.001
SS = sum of squares; MS = mean squares; d.f. = degrees of freedom.
minimum size of the primiparous instar) has been demonstrated by Ebert (1992) in
the same Daphnia species.
A significant interaction between food concentration and oxygen concentration
was detected in body length at the sixth instar, size at maturity, juvenile growth rate
andfirstclutch size, and a nearly significant interaction (P = 0.067) was obtained in
maturation time (Table II). These results demonstrate synergism between the
effects of the two independent variables.
Filter-screen area
The filter-screen areas of Daphnia increased with body size (Figure 2, Table III).
Both food concentration and oxygen concentration had significant effects on the
filter-screen area (Table IV). Reduced food level induced the development of
large filter screens in Daphnia grown at low or high oxygen concentrations. The
filter-screen areas of Daphnia exposed to the low-food condition under low oxygen were significantly smaller than those exposed under high oxygen (ANCOVA,
d.f. = 1,28, F = 39.96, P < 0.001, for intercept), indicating that the low oxygen concentration reduced thefilter-screenarea at the low food level. In other words, the
761
T.Hanazato
Table III. Regressions of the filter-screen area of the third limb (A, mm2) on body length (L, mm) of
D.magna under different food and oxygen conditions Regression model: A = a + bL. Abbreviations of
treatments are the same as in Table I
Treatment
N
a
b
r
P
HfHox
LfHox
HfLox
LfLox
15
15
15
15
-0.0886
-0.1215
-0.0855
-0.1030
0.1128
0.1593
0.1058
0.1382
0.860
0.961
0.881
0.964
<0 001
<0.001
<0.001
<0.001
low oxygen interfered with the development of large filter screens in Daphnia at
the low food level. The effect of low oxygen concentration on filter-screen size was
not significant when the food level was high, however (ANCOVA, d.f. = 1,28,
F= 3.32, P = 0.08).
Discussion
The animal's growth was suppressed by low oxygen concentration. The retarded
growth rate with low oxygen has also been demonstrated in D.magna (Homer and
Waller, 1983) and Daphnia pulex (Hanazato and Dodson, 1995).
Daphnia magna matured at instar 6 (hadfivejuvenile instars) with high oxygen
concentration in the present experiments. The instar number is larger by one than
that of D.magna developed by Ebert (1991). Ebert (1991) has reported that
females which were smaller at birth take more instar numbers during juvenile
stages. This might be why D.magna had a larger juvenile instar number in the present experiments, where small neonates (obtained from thefirstbrood) were used.
The filter-screen areas of Daphnia exposed to the low-food condition under low
oxygen were smaller than those exposed under high oxygen in the present study.
The results support the hypothesis that low oxygen concentration affects the
development of large filter screens in food-limited environments. This effect may
be caused by Daphnia changing its energy allocation. Low oxygen concentrations
lower Daphnia's metabolic rate (Kobayashi and Hoshi, 1984; Weider and Lampert, 1985), thus reducing the amount of energy available for filter-screen development. Low oxygen levels induce hemoglobin production in Daphnia. This may also
be a factor reducing the energy available to develop large filter screens because
Table IV. Two-way ANCOVA for testing the effects of food concentration and oxygen concentration
on the filter-screen area of D.magna
Factor
SS
d.f.
MS
F
P
Food(F)
Oxygen (O)
Interaction (F x O)
Error
Total
0.032%
0.00321
0.00035
0.00867
0.04516
1
1
56
55
58
0.03296
0.00321
0.00000
0.00016
209.20
20.35
0.04
<0.001
<0.001
1.000
SS = sum of squares; MS = mean squares; d.f. = degrees of freedom.
762
Effects of food shortage and O , deficiency on Daphnia
A
A
A
0.2
HfHox
o
LfHox
A
A
HfLox
A
A
LfLox
2 0.16
A
A
c
A
A
Ao
«
A
o
? 0.12
O
A
O
O
o
0.08 •
1
s
1
1
1
1.5
2
2.5
3
Body length (mm)
Fig. 2. Filter-screen areas of the third limbs of different sized D.magna reared with high food-high
oxygen concentrations (HfHox), low food-high oxygen concentrations (LfHox). high food-low oxygen concentrations (HfLox) and low food-low oxygen concentrations (LfLox).
hemoglobin production has a cost in terms of the animal's total energy intake
(Weider and Lampert, 1985).
Morphological changes in elements of filtering limbs other than the filter screens
were not analyzed. One might expect a possibility that some of the elements are
enlarged to increase the area of gas exchange under the low-oxygen condition, so
that energy and materials used for developing the large filter screen are reduced.
This could be a future research subject. However, Dodson and Frey (1989) have
mentioned that gas exchange probably takes place across the entire surface of the
animal, but not at the branchial sacs and not particularly at the thoracic legs.
The present study has clearly demonstrated synergistic effects of food shortage
and oxygen deficiency on Daphnia life history characteristics. Food shortage
reduces energy uptake in Daphnia and increases relative energy loss by respiration
(ratio of respiration to food consumption) (Richman, 1958; Lei and Armitage,
1980). Oxygen deficiency also reduces energy uptake and alters energy allocation
as mentioned above, and thus reduces energy available for growth and reproduction. These changes may be a mechanism causing the synergism.
The effect of low oxygen concentration on Daphnia filter screens is probably
another mechanism for the synergistic effects of the two factors on Daphnia life
history. Oxygen deficiency interferes with the development of large filter screens
under low-food conditions. Filter-screen area has a positive correlation with filtering rate (Lampert, 1994). Thus, the smaller filter-screen area induced by the
oxygen deficiency may reduce thefilteringrate and thus reduce energy uptake by
763
T.Hanaiato
Daphnia. Therefore, energy uptake seems to be reduced by food shortages and
oxygen deficiencies jointly.
This conclusion is contrary to the results of Kring and O'Brien (1976), who demonstrated that D.pulex increasedfilteringrate with increasing hemoglobin concentration in the daphnids. However, they acclimated the animals to the experimental
food concentration (most probably lower than the incipient limiting level of food)
for only 1 h or less in the experiments, which must be too short for the daphnids to
develop largefilterscreens (Lampert, 1994). This suggests that thefilteringrates of
D.pulex given by Kring and O'Brien are lower than those of the same Daphnia
species exposed to the experimental condition for longer periods, and that the
relationship between oxygen concentration and filtering rate in the latter Daphnia
differs from that given by Kring and O'Brien. Thus, the relationship should be
tested again using daphnids satisfactorily acclimated to the food-limited condition.
Lampert (1994) has emphasized the significance of Daphnia's phenotypic plasticity infilter-screenarea with changing food concentration in understanding their
grazing rates. The present study has shown that some environmental stresses, such
as low oxygen concentration, could also be factors affecting filter-screen area.
Thus, this effect should be included in consideration of daphnid feeding ecology.
Low-oxygen layers often develop in the hypolimnion in eutrophic lakes. The
hypolimnion is also a water layer where food for zooplankton is limited. Therefore, Daphnia, by performing diel vertical migration, should often experience the
two environmental stresses—food shortage and oxygen deficiency—together
(Hanazato et al., 1989). Hence, the population dynamics of such Daphnia may be
affected synergistically by the stresses. Furthermore, vertically migrating Daphnia
are likely to simultaneously experience other enviornmentaJ stresses, such as predator kairomones and pesticides, which also have synergistic effects on Daphnia
(Hanazato and Dodson, 1995). Diel vertical migration gives Daphnia the benefit of
reduced mortality due to fish predation, but at the cost of negative synergistic
effects of various environmental stresses.
Acknowledgements
I thank Dr Akio Furukawa, who kindly allowed me to use his planimeter for the
measurement of Daphnia filter-screen area. I am also grateful to Dr Rick Weisburd for helpful comments and linguistic improvements, and to two anonymous
reviewers for improving the paper.
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Received on October 26, 1995; accepted on December 22, 1995
765