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SHORT COMMUNICATION
Tolerance to low ambient calcium
shows inter-population differences in
Daphnia galeata
NINA ALSTAD RUKKE
UNIVERSITY OF OSLO, DEPARTMENT OF BIOLOGY, DIVISION OF LIMNOLOGY, PO BOX
CORRESPONDING AUTHOR:
, BLINDERN,  OSLO, NORWAY
[email protected]
The tolerance to low ambient calcium (Ca) was assessed in two populations of Daphnia galeata.
There was a threshold for survival between 0 and 2 mg Ca l–1. However, the ability to cope with
low Ca concentrations clearly differed between the two populations, as the population from the lowCa locality was less tolerant to low Ca. Additionally, neonate individuals had poorer survival than
adults when reared at ~0 mg Ca l–1, supporting a suggested juvenile bottleneck regarding the tolerance to low Ca concentrations. The mean specific Ca content in adult D. galeata was not different
between the two populations, but individuals reared in medium with 1 mg Ca l–1 only had two-thirds
of the Ca content of those reared in medium with 10 mg Ca l–1. The significant differences between
the two populations investigated suggest that inter-population variation in tolerance to low ambient
Ca concentrations might be important to explain the success of Ca-demanding crustaceans in softwater lakes.
Low levels of calcium (Ca) in softwater lakes may limit the
distribution, growth and reproduction of Ca-demanding
crustacean zooplankton (Tessier and Horwitz, 1990;
Hessen et al., 1995, 2000). Ionic Ca is the main source of
Ca for zooplankton (Marshall et al., 1964; Cowgill, 1976),
and most of the crustacean Ca is present as carbonate and
phosphate minerals in the carapace (Stevenson, 1985). As
zooplankton generally store negligible amounts of Ca
during the moult (Turpen and Angell, 1971; Alstad et al.,
1999), these animals have a recurring need for Ca to complete postmoult calcification of their carapace.
In a survey of 1500 Norwegian lakes, the median Ca
concentration was 1 mg l–1 (Skjelkvåle et al., 1997), while
the corresponding global mean is 15 mg l–1 (Wetzel, 1975).
The deposition of anthropogenic SO42– has decreased
across large proportions of Europe and North America
throughout the 1980s and 1990s (Stoddard et al., 1999).
This decrease, in turn, led to decreased mineral weathering rates and depletion of Ca in watershed soil pools, and
a probably long-lasting depletion of Ca is currently
© Oxford University Press 2002
observed in several acidified areas dominated by softwater
lakes (Lawrence et al., 1999; Stoddard et al., 1999). This
might affect zooplankton community structure in softwater lakes covering large areas like the Canadian Shield
(Yan et al., 1989) and major parts of Scandinavia.
Previous studies have demonstrated incomplete postmoult calcification and reduced survival, growth and
reproduction in Daphnia magna reared in media with low
Ca concentrations (<5 mg Ca l–1) (Alstad et al., 1999;
Hessen et al., 2000). Daphnia magna commonly occur in
hardwater ponds, however, and may thus not be representative of other Daphnia species. In order to test the generality of the observations on D. magna, the present study
assessed the tolerance to low ambient Ca in the related
species Daphnia galeata.
Daphnia galeata is known to occur in softwater lakes with
Ca concentrations down to 0.8 mg l–1 and is thus not
among the most Ca-demanding Daphnia species [data
from Hessen et al. (Hessen et al., 1995)]. However, D. galeata
is reported to be sensitive to low pH levels (Havens, 1992;
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Yan et al., 1996), and this probably excludes the species
from many acidified lakes. As lake pH and Ca concentration are often correlated, laboratory experiments are
needed to separate the effects of these two variables and
assess the Ca threshold for survival of D. galeata. Additionally, little is known about the magnitude of interpopulation differences in the zooplankton response to low
Ca concentrations, although this might be important to
the regional distribution of a species.
This study used an experimental approach to assess the
threshold for survival and the Ca demand of two populations of D. galeata. Artificial test media were used in
the experiments, and this allowed for a separation of
the effects of low water Ca concentrations from other
parameters that usually correlate with Ca in nature, such
as pH, magnesium and other major ions.
Two populations of D. galeata were isolated from the
lakes Erken (>10 mg Ca l–1) and Ånnsjön (2–3 mg Ca l–1).
The animals were kept at 18 ± 1ºC in a thermostatically
controlled room with dim artificial light for 4 months prior
to the onset of the experiments. They were reared in artificial Elendt M7 medium [Organization for Economic
Co-operation and Development (OECD), 1997] with a
Ca concentration of 10 mg l–1, because this level is sufficient for complete postmoult calcification and good survival and growth in the Ca-demanding species D. magna
(Alstad et al., 1999; Hessen et al., 2000). The Elendt M7
medium was made by adding salts and vitamins to distilled
water according to OECD guidelines (OECD, 1997). In
the experiments, the Ca level of the media was manipulated to nominal concentrations of 0, 1, 2, 5 and 10 mg l–1
(measured concentrations after addition of food were 0.5,
1.3, 2.2, 5.5 and 10.3 mg l–1). All other elements were kept
constant, and the pH was adjusted to 7.8 ± 0.2 by addition
of HCl or NaOH. Manipulation of the Ca concentration
of the medium produced only small changes in ionic
strength (50–55 l0–4 M) and conductivity (217–273 µS
cm–1), and thus effects caused by differences in ionic
strength or osmoregulation ability were probably small.
The animals were fed a culture of the green alga
Selenastrum capricornutum. To reduce contamination of Ca
from the algal medium to the zooplankton culture and
ensure similar food quality during the experiments, a
dense culture of Selenastrum was washed twice with Ca-free
medium and frozen in small batches at –20ºC. The
animals were fed every third day, giving a final concentration of 0.9 mg C l–1 (0.75 mM C) in the cultures. This
should be well above the incipient limiting level of 0.2–0.5
mg C l–1 (Lampert, 1987).
In the first experiment, the survival of neonate (<24 h
old) and adult D. galeata was assessed in medium with
nominal Ca concentrations of 0, 2 and 5 mg l–1. Prior
to the experiment, the animals were rinsed in Ca-free
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medium and randomly divided among the different Ca
concentrations. For both neonate and adult animals, each
Ca concentration and population was represented with 10
beakers with 50 ml of medium, each beaker containing
five individuals. Survival was recorded every day, and
dead animals were removed. The experiment lasted for
7 days to ensure that all animals had completed at least
one moult. Under similar conditions, intermoult periods
of D. magna are found to vary from 2 days in neonates to
4–5 days in adult animals (Hessen et al., 2000).
In the second experiment, the animal specific Ca content
(i.e. Ca as a percentage of dry weight) was measured. Adult
animals from both populations were rinsed in Ca-free
medium and randomly divided among media with nominal
Ca concentrations of 1, 2, 5 and 10 mg l–1. Each Ca
concentration and population was represented with three
beakers containing 1 l of medium and 35 animals. After 7
days, the animals were rinsed in distilled water and individual lengths were measured. Dry weights were calculated
using the length/dry weight relationship for D. galeata from
Bottrell et al. (Bottrell et al., 1976), assuming a fixed
length/weight ratio. The animals were dried at 60ºC for 24
h and cooled in a desiccator. Each sample containing
35 animals was digested in 0.1 ml of 50% HNO3 at 120ºC
for 30 min. Ca content was analysed on a Varian
SpectrAA 10 atomic absorption spectrophotometer using a
N2O–acetylene flame. A 5000 µg ml–1 KCl solution was
added to avoid ionization of Ca.
The statistical analyses were performed with the JMP
3.2.2 computer program (Statistical Analysis Systems
Institute, 1997). As a linear relationship between the Ca
concentration in the test medium and animal Ca content
could not be assumed, analyses of variance (ANOVA)
were performed using Ca concentration as an ordinal
variable. Tukey–Kramer HSD was performed to test for
significant differences between groups.
All the neonates from the Ånnsjön population reared in
medium with 0 mg Ca l–1 died within 2 days (Figure 1A).
However, 80% of the neonates from the Erken population
were still alive after 4 days, and during the rest of the
experiment there was no mortality. Regarding the adults,
40% of the individuals from the Ånnsjön population
reared in medium with 0 mg Ca l–1 died during the first
3 days, while all the adults from the Erken population survived the whole experimental period (Figure 1B). There
was no mortality among neonates or adults reared in
medium with 2 and 5 mg Ca l–1.
In both populations, the mean specific Ca content of
adult D. galeata was significantly affected by the Ca
concentration of the medium (one-way ANOVA; Erken:
F[3,8] = 46.49; P < 0.0001; Ånnsjön: F[3,8] = 25.76; P =
0.0002) (Figure 2). Individuals reared in medium with
1 mg Ca l–1 had only two-thirds of the Ca content of those
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Fig. 1. Survival (± SD) of (A) neonate and (B) adult D. galeata from the lakes Erken () and Ånnsjön () reared in medium with 0 mg Ca l–1.
n = 10 beakers for each population; each beaker contained five individuals.
reared in medium with 10 mg Ca l–1. However, there was
no significant difference in Ca content between the two
populations (one-way ANOVA, P > 0.3137). Additionally,
there were no differences in individual dry weight (mean
± SD = 14.9 ± 2.57 µg) between animals from different
populations or medium Ca concentrations (two-way
ANOVA, all P > 0.05, interaction tested). This result is
important in the present context because weight-specific
Ca content generally decreases with animal size in crustaceans (Porcella et al., 1969; Huner and Lindqvist, 1985;
Alstad et al., 1999; Alstad Rukke, 2002).
The threshold Ca concentration for survival of
D. galeata appeared to be between 0 and 2 mg l–1, depending on the population. The two populations clearly differed
in their tolerance to low Ca concentrations. Somewhat surprisingly, the population from the low-Ca locality was least
tolerant to low Ca. Because of the poor survival of individuals from lake Ånnsjön at 0 mg Ca l–1, it was not
possible to measure the Ca content of these animals.
Nevertheless, as the two populations had similar Ca contents when reared at sufficient Ca concentrations, they presumably also had similar Ca demands. The difference in
Fig. 2. Mean specific Ca content (± SD) as a percentage of dry weight in adult D. galeata from (A) Lake Erken and (B) Lake Ånnsjön reared for 7
days in medium with different Ca concentrations (nominal values). n = 3 samples for each concentration; each sample contained 35 animals.
Different letters above figure bars denote significant (P < 0.05) differences between treatments (Tukey–Kramer HSD).
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survival at low Ca concentrations may thus be a result of
different affinity of Ca uptake mechanisms. A significant
proportion of the Ca uptake in crustaceans is known to
occur by active transport (Marshall et al., 1964; Wright,
1979). Additionally, Flik et al. demonstrated both an ATPdependent Ca pump and a Na+/Ca2+ exchanger in the
crab Carcinus maenas (Flik et al., 1994). However, there is
little information on the ability of crustaceans to adapt to
low ambient Ca concentrations by increasing the efficiency of Ca uptake, although freshwater crayfish generally have uptake mechanisms with higher affinities than
marine species (Neufeld and Cameron, 1993).
Inter-population differences in the threshold Ca concentration for survival are also observed in Gammarus spp.
(Vincent, 1969; Økland and Økland, 1985). Vincent
(Vincent, 1969) demonstrated a higher threshold level in
a hardwater population compared to a softwater population of Gammarus pulex, while a large regional survey by
Økland and Økland (Økland and Økland, 1985) showed
that lowland populations of Gammarus lacustris had a
higher Ca threshold for survival than populations from
cold mountain lakes. However, these studies provide no
physiological explanation of the observed pattern, and the
correlation between parameters important to Ca uptake,
like water Ca, pH, Na, HCO3– and temperature, often
makes it difficult to suggest a causal factor on the basis of
presence/absence data only. In the present study, the
trend that populations native to softwater lakes tolerate
low ambient Ca better was reversed, as the D. galeata population from the lake with highest Ca concentrations had
the lowest Ca threshold. It is thus necessary to assess
experimentally the Ca threshold of a large number of
populations, or even clones, to reveal a possible causative
mechanism explaining inter-population differences in the
tolerance to low Ca concentrations.
The survival of neonate D. galeata reared in medium
with 0 mg Ca l–1 was found to be lower than that of adults
reared in the same medium. Corresponding results are
found for G. lacustris (Alstad Rukke, 2002). In addition,
neonate individuals of several crustacean species are
found to have a higher specific Ca content than adults
(Porcella et al., 1969; Huner and Lindqvist, 1985; Alstad et
al., 1999; Alstad Rukke, 2002), and hence they probably
also have a higher Ca demand. This led Hessen et al. to
suggest that early juvenile crustaceans experience a bottleneck regarding the susceptibility to Ca deficiency
(Hessen et al., 2000).
In the present study, adult D. galeata reared in medium
with 10 mg Ca l–1 had a specific Ca content of 2.7%. This
is considerably lower than the Ca content of adult D.
magna (4.2%) (Alstad et al., 1999), but comparable to that
of Daphnia tenebrosa (2.3%) (Hessen and Alstad Rukke,
2000) reared under similar conditions. Nevertheless,
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Daphnia spp. in general apparently have high Ca contents
(Cowgill, 1976; Havas, 1985) compared to other cladoceran species like Holopedium gibberum (0.2–0.5%),
Diaphanosoma brachyurum (0.2%) and to copepods (0.06%)
(Yan et al., 1989). Daphnia galeata reared in medium with Ca
concentrations <5 mg l–1 contained less Ca than individuals reared at higher Ca concentrations, and such
incomplete calcification has been shown to be associated
with reduced longevity, growth and egg production in D.
magna (Alstad et al., 1999; Hessen et al., 2000). Hence the
Ca threshold for long-term persistence of populations
might be higher than the recorded threshold for survival
during one moult.
Daphnia galeata was found to have a relatively high Ca
demand, in line with what is known about other species of
the Daphnia genus. However, the two populations showed
pronounced differences in their tolerance to low Ca concentrations, as the population native to the lake with the
lowest Ca concentration had the poorest survival at 0 mg
Ca l–1. As the isolated laboratory populations only represented a subsample of the native populations, the mean
performance of the laboratory and native populations
might be somewhat different. Thus, possible intra-population differences together with the inter-population
differences shown demonstrate the necessity to assess the
performance of several clones and populations in order to
decide whether or not a species is negatively affected by
low Ca concentrations.
AC K N O W L E D G E M E N T S
The author is grateful to Tobias Vrede for providing the
two populations of D. galeata. I also want to thank Bjørn
Arne Rukke for assistance during the experiments, and
Dag O. Hessen, Anders Hobæk, Ian Jenkinson, Bjørn
Arne Rukke, Asbjørn Vøllestad and an anonymous
referee for constructive comments on the manuscript.
This study was financed by the Norwegian Research
Council (NFR), grant no. 113765/410.
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