Effects of hypoxia acclimation on morpho-physiological traits over three generations of Daphnia magna
Institut für Zoophysiologie, Westfälische Wilhelms-Universität, Hindenburgplatz 55, 48143 Münster, Germany
* Author for correspondence (e-mail: pirow{at}uni-muenster.de)
Accepted 22 March 2005
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Summary |
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Key words: Crustacea, Branchiopoda, Daphnia magna, acclimation, circulation, growth, hypoxia, haemoglobin, NADH, oxygen transport, respiration, ventilation
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Introduction |
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Given these constraints on the oxygen transport systems, the question
arises as to how filter-feeding zooplankters can ensure a certain degree of
oxygen homeostasis under a wide range of
PO2amb. Oxygen homeostasis here refers to the
physiological ability to minimize environmentally induced
perturbations in internal (steady-state) oxygen levels while ensuring an
adequate flow of oxygen to the tissues. It provides the twofold advantage of
maintaining aerobic metabolism (as the most efficient mode of fuel
utilization) and minimizing the risk of oxidative stress to tissues. There are
various ways of controlling perturbations in biological systems (cf.
Jones, 1973). Recent studies
on the euryoxic zooplankter Daphnia magna have revealed two
principally different regulatory mechanisms, negative feedback and buffering.
Negative feedback is inherent in the circulatory and ventilatory systems as
reflected by the inverse relationship between heart/limb beating rate and
PO2amb
(Paul et al., 1997
;
Pirow and Buchen, 2004
).
`Oxygen buffering' (Jones,
1972
) refers to the stabilizing effect occurring when haemoglobin
(Hb) present in high concentration is reversibly loaded and unloaded along the
steep part of its oxygen equilibrium curve
(Pirow, 2003
;
Pirow et al., 2004
).
Chronic exposure of D. magna to environmental hypoxia induces gene
expression-mediated adjustments in Hb concentration and oxygen affinity
(Zeis et al., 2003a). This
acclimatory response improves the quality of oxygen regulation under
conditions of protracted ambient oxygen deficiency
(Pirow et al., 2001
). There
are indications that hypoxia acclimation in Daphnia sp. causes
additional changes in other characteristics such as metabolic rate (fig. 5 of
Kobayashi and Hoshi, 1984
;
Wiggins and Frappell, 2000
)
and body size (Kobayashi,
1982
), which may contribute to an improved hypoxia tolerance. The
aim of the present study is to investigate the full spectrum and the extent of
hypoxia-induced acclimatory changes by measuring all morpho-physiological
parameters relevant in oxygen transport and regulation. In addition, the
dynamics and possible transgenerational effects of hypoxia acclimation were
studied. Finally, we tested whether the transition to and maintenance of the
low-oxygen acclimation state is associated with costs reducing the
reproductive success of the animal.
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Materials and methods |
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Selection of animals and experimental schedule
To obtain synchronized clonal animals, 26 parthenogenetic offspring of the
third brood of a single female were raised under normoxic conditions. The
third brood of these offspring was selected for the experiments and
represented the parental (P) generation. The P generation was raised in
several batches of 70-80 animals under normoxic conditions. When the first
eggs appeared in the brood chamber at age 8 days, the P generation was divided
and exposed to the two acclimation conditions
(Fig. 1). Starting at age 8
days, batches of 8-15 individuals were successively sampled from each
acclimation group in the initial phase (i.e. within the first third) of each
of the first five reproductive cycles for the measurements. A sixth
measurement followed some reproductive cycles later. A single reproductive
cycle was regarded to be initiated by the release of eggs into the brood
chamber. The first (F1) and second filial (F2)
generation were derived from the third brood of the respective parental
generation.
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Sampled females carried parthenogenetic embryos of developmental stage 1-2
(Green, 1956). The selection
of females according to the developmental stage of their embryos ensured that
the animals were always in the same stage of their moulting cycle (i.e. within
24 h after ecdysis). The embryos were removed by flushing the brood chamber
with a thinly drawn-out glass capillary. The number of embryos was counted,
and the carapace length (Lc, mm) of the mothers was
measured as the distance between the anterior and posterior carapace margin.
In addition, the distance between the base of the apical spine and the
anterior part of the head was determined to derive a relationship between
Lc and body length Lb
(Lb=1.108Lc+0.157,
r2=0.99, N=341). Lc was
further used derive the dry mass (W, µg) according to the
relationship lnW=3.05+2.16lnLc
(Kawabata and Urabe, 1998
).
The debrooded mothers were then kept in nutrient-free culture medium or
respiratory medium (see below) for 2 h before starting the physiological
measurements.
Of a sampled batch of 8-15 individuals, 3-4 animals were used in a single respiration measurement, 3-4 animals were used to obtain one pooled sample of haemolymph for the determination of Hb concentration and oxygen affinity, and two individuals were used to analyse the ventilatory-circulatory characteristics.
Respiration measurements
Oxygen consumption rate of a group of 3-4 animals was measured at
20.0±0.1°C in a 1.5 ml closed respirometer (DW1; Hansatech
instruments, Reutlingen, Germany). The respiratory medium consisted of
M4-medium containing 12.75 mmol l-1 Hepes-buffer (pH
8.0±0.1) as well as Tetrazyclin and Streptomycin (12.5 mg
l-1 each). The chamber was cleaned every day using 70% ethanol. For
calibration, the medium in the chamber was equilibrated with air and
normocapnic nitrogen. The respiratory activity of the animals and dependence
on PO2amb was determined by monitoring the
decline in oxygen concentration from normoxia to anoxia. At the end of the
experiment, animals were removed without exchanging the chamber medium, and a
second calibration and blank measurement followed. The oxygen consumption rate
at different PO2amb values was obtained from
small decrements in oxygen concentration per increment in time. After taking
the blank measurement into account, the mass-specific oxygen consumption rate
(O2, nmol
h-1 mg-1) was obtained by dividing the whole-animal
oxygen consumption rate
(
O2, nmol
h-1) by dry mass of the animals.
Concentration and oxygen affinity of haemoglobin
A pooled haemolymph sample (1.5-2.5 µl) was drawn from 3-4 animals as
previously described (Pirow et al.,
2001) and aspirated into a glass capillary (inner and outer
diameter: 0.3 mm i.d., 0.7 mm o.d.; Hilgenberg, Malsfeld, Germany). The
capillary was transferred into the light path of a monolithic miniature
spectrometer (MMS-UV/VIS, spectral range: 194-738 nm, 256 pixel photodiode
array; Carl Zeiss, Oberkochen, Germany). The spectrometer was connected
via Front End Electronics and a PC interface board (14 bit
resolution; tec5 AG, Oberursel, Germany) to a computer
(Becher, 2002
). Absorption
spectra (520-590 nm) were acquired (SDAS_32D software, tec5 AG) under
oxygenated conditions using water as reference. The haem-based Hb
concentration was determined according to Lambert-Beer's law taking an
appropriate correction for the optical path length of the cylindrical
capillary into account (Becher,
2002
).
Oxygen equilibria were determined on 1 µl haemolymph samples in a
diffusion chamber linked to mass flow controllers (Tylan FC280/FC260; General
TCA GmbH, Eching, Germany). The chamber was positioned in the light path of
the miniature spectrometer. A drop of silicon oil (AK 350; Wacker Silicone,
München, Germany) covered the sample to avoid dehydration. The sample was
equilibrated with gas mixtures of 12 different oxygen tensions
(Fig. 4F), which were obtained
by mixing air with normocapnic nitrogen. The oxygen tension was checked by an
oxygen analyzer (S3A-II Ametek; Thermox instruments division, Pittsburgh,
USA). Oxygen saturations were calculated from the absorption spectra as
previously described (Pirow et al.,
2001). The half-saturation oxygen tension
(P50) and Hill's cooperativity coefficient were obtained
by fitting a sigmoid curve to the oxygenation data using the Hill equation
(Stryer, 1995
).
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Statistics
Unless otherwise stated, data are expressed as mean values ±
standard deviation (S.D.) with N indicating the
number of animals examined. Differences in the body size-dependence of stroke
volume were assessed by an analysis of covariance (ANCOVA). Comparisons
between the two acclimation groups were performed over all generations as well
as within each generation by a nonparametric two-sample rank test
(Mann-Whitney test). Within each acclimation group, intergenerational
differences were checked by an analysis of variance by ranks (Kruskal-Wallis
test). In the case of a statistical significant difference, multiple
comparisons among pairs of rank sums (Dunn's test) were used to determine
between which generations the differences existed. Linear regression analysis
(Zar, 1999) was used to assess
the body size-dependence of a morpho-physiological variable. Statistical
differences were considered as significant at P<0.05. All
statistical analyses were performed using SigmaPlot (version 8.0; SPSS Inc.),
SigmaStat (version 3.01; SPSS Inc.) and Statistica (version 6.0; Statsoft
Inc.).
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Results |
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Body growth and clutch size
The carapace length increased with age (7-37 days) and ranged from 1.9 to
3.4 mm (Fig. 2). The influence
of hypoxia acclimation on body growth was assessed by calculating the relative
difference in the carapace length (Lc) of both acclimation
groups for each of the six reproductive cycles. These relative differences
were then averaged. Hypoxia acclimation resulted in a reduction of
Lc by 0.9±3.3% (mean ±
S.D. of six reproductive cycles) in the P generation, by
11.1±2.6% in the F1 generation, and by 9.7±3.3% in
the F2 generation (Fig.
2G-I). The number of embryos per brood increased with
Lc without showing any clear influence of hypoxia
acclimation (Fig. 2D-F). The
cumulative number of eggs per animal during the first five reproductive cycles
was 77 and 74 (normoxia-acclimated vs hypoxia-acclimated) in the P
generation, 62 and 56 in the F1 generation, and 61 and 57 in the
F2 generation. The reproductive success of the
F1/F2 generations was accordingly lower than that in the
P generation.
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Concentration and oxygen-binding properties of haemoglobin
The haem-based Hb concentration increased by 266% as a consequence of
hypoxia acclimation (Table 1).
Significant differences between both acclimation groups occurred in all
generations (Fig. 4B). In
addition, the half-saturation oxygen tension (in vitro
P50) decreased by 32%
(Table 1;
Fig. 3F). Again, differences
between both acclimation groups occurred in all generations
(Fig. 4C). Intergenerational
differences were only detectable in the hypoxia-acclimated group
(Kruskal-Wallis test: P=0.02). Multiple comparisons revealed a
difference between the P and the F2 generation (Dunn's Method:
P<0.05; Fig. 4C).
The Hill's cooperativity (n50) coefficient decreased by 8%
as a result of hypoxia acclimation (Table
1).
In vivo oxygenation of Hb
The ambient oxygen tension effecting a half saturation of Hb in the heart
region (in vivo P50) decreased by 35% as a consequence of
hypoxia acclimation (Table 1,
Fig. 3E). Significant
differences between both acclimation groups occurred in all generations
(Fig. 4K).
Appendage beating rate
Exposing the two acclimation groups to normoxic conditions revealed a 19%
lower appendage beating rate (fA) in the
hypoxia-acclimated group (Table
1; Fig. 3C).
Significant differences between both acclimation groups occurred in the
F1 and F2 generations
(Fig. 4D). Intergenerational
differences were only present in the normoxia-acclimated group (Kruskal-Wallis
test: P<0.01). Multiple comparisons revealed a significant
difference between the P and F2 generations (Dunn's Method:
P<0.05). The exposure to a gradual transition from normoxia to
anoxia did not induce a counteracting response in fA (Figs
3C,
4E), except for the
hypoxia-acclimated animals, which showed a slight hyperventilation below
2 kPa with a maximum fA at 0.7 kPa.
Heart rate
The exposure to progressive hypoxia induced a counteracting response in
heart rate (fH), indicating the presence of an
oxyregulatory (feedback) mechanism. Its operating range was the same in both
acclimation groups as reflected by the identity of the lower threshold
(PHP=6.1-6.2 kPa; Table
1; Figs 3D,
4I), below which the
fH reached maximal values. Except at extreme values of
PO2amb (1-2 kPa), the fH of
both acclimation groups was essentially the same, indicating the absence of an
acclimation effect (Fig.
4F-H).
Indicators of hypoxia tolerance
The improvement of hypoxia tolerance as a consequence of hypoxia
acclimation was reflected by the reduction of critical ambient oxygen partial
pressures (Pc,O, Pc,A and
Pc,H) at which oxygen consumption rate, appendage beating
rate and heart rate, respectively, started to decrease over-proportionally
(Fig. 3A,C,D). The
Pc,O decreased by 52%
(Table 1). Significant
differences between both acclimation groups occurred in the F1 and
F2 generations (Fig.
4L). The Pc,A decreased by 66%
(Table 1). Significant
differences occurred in all generations
(Fig. 4N). Intergenerational
differences were only present in the normoxia-acclimated group (Kruskal-Wallis
test: P=0.01). The Pc,H decreased by 21%
(Table 1). Significant
differences occurred in the F1 and F2 generation
(Fig. 4J). The improvement of
hypoxia tolerance was also signalized by the 61% reduction of
Pc,N (Fig.
3B, Table 1), the
ambient oxygen partial pressures at which the NADH fluorescence intensity of
the appendage muscles started to increase. This increase indicated the
impairment of tissue oxygen supply (Pirow
et al., 2001). Significant differences occurred in all generations
(Fig. 4M).
Dynamic of hypoxia acclimation
The main acclimatory adjustments occurred in the P generation within the
first 3 days after the start of hypoxic incubation. The fast transition to the
low-oxygen acclimation state is apparent in the Hb concentration and oxygen
consumption rate
(O2) as well as
in the indicators of hypoxia tolerance such as Pc,A and
Pc,N (Fig.
6).
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Body-size dependence
The linear dependence of a physiological variable on carapace length was
tested in both acclimation groups over all generations
(Table 1). As an exception, the
P generation of the hypoxia-acclimated group was excluded because of the
transient acclimatory adjustments. A significant positive dependence was found
for the Hb concentration (hypoxia-acclimated: P=0.04;
Fig. 7A) and the
Pc,H (both acclimation groups: P<0.01;
Fig. 7G). A negative dependence
occurred in the in vitro P50 (hypoxia-acclimated:
P<0.01; Fig. 7B),
the appendage beating rate at the Pc,A (both acclimation
groups: P<0.01; Fig.
7C), the Pc,N (normoxia-acclimated:
P=0.03; Fig. 7D), and
the heart rate at normoxia and at the Pc,H
(hypoxia-acclimated: P<0.01;
Fig. 7E,F).
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Discussion |
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These acclimatory adjustments improved the hypoxia tolerance by reducing
the critical ambient oxygen partial pressures (Pc,O,
Pc,A, Pc,H) at which oxygen
consumption rate, appendage beating rate, and heart rate started to decrease
over-proportionally (Fig.
3A,C,D). The reduction of Pc,H was only 21%
(Table 1). The
Pc,O and Pc,A decreased by 52% and
66%, a change quite similar to the 61% reduction in Pc,N
(Table 1), which signalized the
incipient impairment of tissue oxygen supply
(Pirow et al., 2001).
Of the two regulatory mechanisms, negative feedback and oxygen buffering
(via Hb), only the latter became reinforced during hypoxia
acclimation whereas the former remained unaffected. Confronting differently
acclimated animals with a short-term (80 min) transition from normoxia to
anoxia elicited a counteracting increase in heart rate (compensatory
tachycardia; Paul et al.,
1997
), which indicates the presence of an active negative-feedback
mechanism. Its operating range and the magnitude of fH in
dependence on PO2amb were the same in both
acclimation groups (Fig. 3D),
suggesting a uniform oxygen-dependent drive of heart rate
(Paul et al., 2004
). The
appendage movement, which may assume an oxyregulatory function under food-rich
conditions (Pirow and Buchen,
2004
), was essentially unaffected by decreasing
PO2amb except at extreme values of
2 kPa,
at which hypoxia-acclimated animals developed a slight hyperventilation
(Fig. 3C). These animals, when
tested under normoxic conditions, also had a 19% lower appendage beating rate
compared to the normoxia-acclimated group. It seems, however, unlikely that a
reduction of that magnitude has a significant effect on internal oxygen levels
as control analysis has shown (Pirow and
Buchen, 2004
).
The oxygen-buffering mechanism was reinforced during hypoxia acclimation by adjustments at the Hb level (see below). This mechanism comes into play when the internal oxygen levels decrease to such low values that Hb is reversibly loaded and unloaded along the steep part of its oxygen equilibrium curve. As signalized by the changes in Hb oxygen saturation in the heart region (Fig. 3E), this condition (inside the animal) became satisfied when the PO2amb approached and fell below the lower threshold of the circulatory feedback mechanism (PHP=6.1-6.2 kPa, both acclimation groups). Hypoxia acclimation broadened the operating range of the oxygen-buffering mechanism by reducing the lower threshold (Pc,O) from 4.3 to 2.0 kPa. This gain of operating range is almost congruent with the range of low-oxygen conditions (PO2amb=3.9-2.1 kPa) to which the animals became acclimated. The adjacency of the operating ranges for oxygen buffering via Hb (2-6 kPa) and circulatory control (6-20 kPa) is an interesting example for the transition of regulatory control from one mechanism to another, which allows the animal to extend the range of tolerable ambient oxygen tensions.
Comparison with previous data
The haem-based Hb concentrations of normoxia-acclimated and
hypoxia-acclimated D. magna (130 vs 476 µmol
l-1) are comparable to previous data (49-114 µmol l-1
vs 337-600 µmol l-1;
Pirow et al., 2001;
Zeis et al., 2003a
). The
in vitro P50 (1.01-1.20 kPa) measured in whole-blood
samples of the normoxia-acclimated generations was similar to the value of 1.0
kPa reported by Zeis et al.
(2003a
). In the
hypoxia-acclimated animals, this variable decreased to 0.92, 0.66 and 0.61 kPa
in the P, F1 and F2 generations. The latter two values
agree with the value of 0.6 kPa from long-term acclimated animals
(Zeis et al., 2003a
).
Subsequent comparisons are restricted to normoxia-acclimated animals, as
most data from literature are available for animals raised under normoxic
conditions. The mass-specific oxygen consumption rate
(O2) of
debrooded, fasting (2 h) animals being in the first third of their
reproductive cycle was 268 nmol mg-1 h-1 under almost
normoxic conditions (PO2amb=18 kPa) and
20°C. Glazier (1991
)
reported a similar value of 234 nmol mg-1 h-1 for
debrooded, fasting (24 h) animals. The dependence of stroke volume on carapace
length followed a power function similar to that given by Bäumer et al.
(2002
).
Upon response to a progressive reduction in
PO2amb, our animals showed the typical
responses in in vivo oxygen saturation of Hb, heart rate and
appendage beating rate (Paul et al.,
1997; Pirow et al.,
2001
; Pirow and Buchen,
2004
). Against our expectation, the critical ambient oxygen
partial pressure of tissue oxygen supply (Pc,N=2.7 kPa)
deviated from that of oxygen uptake rate (Pc,O=4.3 kPa).
The latter values agrees quite well with the Pc,N of 4.6
kPa reported by Pirow et al.
(2001
). The deviation
therefore cannot be explained by the two different experimental conditions,
under which the animals were either able to swim freely in a closed
respirometer or tethered in a perfusion chamber. The lower
Pc,N obtained in the present study was possibly the
consequence of the replacement of the light source used for NADH excitation.
Greater instabilities in light intensity might have impeded the early
detection of the incipient increase in NADH fluorescence intensity under
progressive hypoxia.
Adjustments at the Hb level
The comparison of differently acclimated D. magna revealed the
known negative correlation between Hb concentration and half-saturation oxygen
tension (Table 1;
Kobayashi et al., 1988;
Zeis et al., 2003a
). The
advantage of this inverse relationship is to minimize the total Hb
concentration required to ensure the adequate supply of oxygen to the tissues
under different ambient oxygen conditions
(Kobayashi et al., 1994
).
Hb-poor animals living under moderate-to-high oxygen conditions benefit from a
low-affinity Hb, which operates at a higher and more extended range of
(internal) oxygen levels. Using instead a high-affinity Hb for the same range
of oxygen tension would require a higher concentration to obtain the same
transport/buffering capacity for oxygen. In contrast, Hb-rich animals living
under low ambient oxygen conditions benefit from a high-affinity Hb.
Theoretical considerations based on the analysis of finely-resolved oxygen
equilibrium curves suggest that animals having a low-affinity Hb instead would
need more than three times as much Hb to transport the same amount of oxygen
(Kobayashi et al., 1994
). As
previously discussed (Bäumer et al.,
2002
; Paul et al.,
2004
), the increased oxygen affinity of Hb in hypoxia-acclimated
animals might compromise the unloading of oxygen to the tissues. The reduction
of oxygen consumption rate and body size mitigates this problem by lowering
critical partial pressure needed to drive the diffusion of oxygen into the
tissues.
Dynamic of hypoxia acclimation
The transfer from the normoxic to the hypoxic environment occurred when
females had entered into the first reproductive cycle. Analyzing these animals
3 days later showed a considerably higher concentration of Hb compared to
animals kept in parallel under normoxic conditions
(Fig. 6A). The induction of Hb
in D. magna is a fast process. Transcripts of hypoxia-inducible Hb
genes occur within a few hours after starting the hypoxic incubation
(Zeis et al., 2003a). Initial
differences in Hb concentration are already detectable after 1 day of hypoxic
incubation (Kobayashi et al.,
1990
), and 3 days are required to attain a stable high level
(Kobayashi et al., 1990
;
Zeis et al., 2003b
). The
present study shows that the rapid induction of Hb is paralleled by a rapid
reduction (within 3 days) in oxygen consumption rate
(Fig. 6B). The simultaneous
reduction of tolerance indicators (Pc,A and
Pc,N; Fig.
6C,D) confirm that a 3-day period is sufficient for adults to
transit to a new acclimation state.
Metabolic rate, growth and reproduction
When tested at almost normoxic conditions
(PO2amb=18 kPa), hypoxia-acclimated animals had
a 22% lower (mass-specific) oxygen consumption rate
(O2) compared to
normoxia-acclimated animals. Based on internal oxygen measurements
(Pirow et al., 2004
), it is
reasonable to assume that under these test conditions the internal oxygen
levels were relatively high and comparable in both groups. Accordingly,
cellular oxygen levels cannot be responsible for the low
O2 of the
hypoxia-acclimated group. Changes in metabolic rate might arise from
alterations in mitochondrial density and/or capacity, which are well known to
occur in animals during thermal acclimation
(Pörtner, 2002
). In
D. magna, low internal oxygen levels arising from the chronic
exposure to a hypoxic environment could effect intermediate-term adjustments
in mitochondrial function.
The reduction of
O2 was
correlated with a decrease in somatic growth rate. Hypoxia-acclimated animals
had a smaller body size than similar-aged normoxia-acclimated animals
(Fig. 2G-I). Both acclimation
effects had already become apparent in the P generation a few days after
starting the hypoxic exposure (Figs
2G,A,
6B). The growth retardation was
more pronounced in the F1 and F2 generations, which
experienced the low-oxygen conditions from birth onwards. Smaller body sizes
are advantageous for diffusive oxygen-transport processes and therefore
contribute to an improved hypoxia tolerance
(Pirow and Buchen, 2004
;
Pirow et al., 2004
). In
contrast to somatic growth, the reproductive success remained essentially
unchanged. Hypoxia-acclimated animals, although being smaller than
normoxia-acclimated animals, had similar clutch sizes during the first five
broods (Fig. 2D-F).
A lower O2
implies that less energy is available for maintenance, growth and
reproduction. A significant anaerobic supplementation of energy provision is
unlikely because in Hb-rich animals the increase in the concentration of
anaerobiosis indicators (lactate) occurs at a
PO2amb lower than 2 kPa
(Usuki and Yamagushi, 1979
).
The present study shows that hypoxia-acclimated animals of D. magna
maintain reproductive performance while reducing the energy allocation to
growth-related processes. The experimental data provide no obvious indications
of a reduction of maintenance costs. The appendage beating rates and the
duration of the moulting cycles of both groups (Figs
3C,
1) at the respective
acclimation conditions (2.1-3.9 vs 17.7-20.0 kPa) were essentially
the same, suggesting similar energetic expenditures for both processes.
Intergenerational differences
The parental environment and state can significantly impact offspring
performance. In Daphnia sp., such maternal or transgenerational
effects are described for inducible defences in response to predator-borne
chemicals (Agrawal et al.,
1999) and for resting egg production in response to photoperiod
and food conditions (LaMontagne and
McCauley, 2001
; Alekseev and
Lampert, 2001
). Besides the possibility of transmitting
information about the environment to the progenies, the maternal state itself
can influence the energy allocation to the offspring, thereby altering their
growth rates, survivorship and fecundity
(LaMontagne and McCauley,
2001
).
Following the response of D. magna to a new oxygen environment
over three successive generations revealed intergenerational differences in Hb
oxygen affinity (Fig. 4C),
growth (Fig. 2G-I) and
reproduction (Fig. 2D-F). The
clutch size in the F1/F2 generations was reduced in
comparison to the P generation. However, this intergenerational difference
occurred not only in the hypoxia-acclimated group but also in the
normoxia-acclimated group. Differences in reproductive performance could arise
from the fact that the mothers of the P generation were cultured in batches of
25 animals, whereas the mothers of the F1 and F2
generations were raised in batches of 70-80 individuals (for crowding effects,
see Guisande, 1993;
Goser and Ratte, 1994
).
Body size and Hb oxygen affinity were affected by hypoxia acclimation, and
both variables again assumed lower values in the F1/F2
generation (Figs 2G-I,
4C). These intergenerational
differences, however, resulted mainly from differences in the onset and
duration of hypoxic exposure during ontogenesis. The
F1/F2 generations experienced the low oxygen condition
from birth onwards, whereas the P generation became exposed to hypoxia after
reaching maturity. The growth retardation was correlated with a reduction of
O2, which
occurred in all hypoxia-acclimated generations. The higher in vitro
P50 of the P generation can be explained by the mixing of
newly synthesized Hb species of high affinity with low-affinity species
(Zeis et al., 2003a
) that were
already present in juvenile stages. Individuals of the
F1/F2 generations, in contrast, produced high-affinity
haemoglobins from birth. In conclusion, a hypoxia-induced transgenerational
effect responsible for the observed intergenerational differences cannot be
substantiated.
Body-size dependencies
In addition to the whole body oxygen consumption rate
(O2) and clutch
size, several other variables were correlated with body size, either in one or
both acclimation groups. The Hb concentration and oxygen affinity of
hypoxia-acclimated animals increased with body size
(Fig. 7A,B), thereby
counteracting growth-related problems in internal oxygen transport. Higher
concentrations of Hb are associated with an increase in haemolymph viscosity
and oxygen-transport capacity, which might explain the negative correlation
between heart rate and body size in hypoxia-acclimated animals
(Fig. 7E,F). In both
acclimation groups, appendage beating rate at the Pc,A
decreased with body size, indicating that larger animals were unable to move
the limbs at the same maximum frequency as smaller ones. Both acclimation
groups also showed a positive correlation of the Pc,H with
body size (Fig. 7G). The supply
of oxygen to the heart at critical PO2amb
therefore seems more dependent on body size than on Hb concentration.
Synopsis
Heart rate control and oxygen buffering via Hb are the main
oxyregulatory mechanisms that enable Daphnia magna to respond
immediately to fluctuating ambient oxygen levels. Conditions of protracted
ambient oxygen deficiency induce intermediate-term adjustments at the Hb and
metabolic levels but none at the systemic level. The main acclimatory
adjustments are completed within 3 days after starting the hypoxic exposure.
The expression of hypoxia-inducible Hb genes results in an increase in the
concentration and oxygen affinity of Hb. The decrease in mass-specific oxygen
consumption rate, which might result from mitochondrial adjustments, reduces
the energy allocation to somatic growth without greatly affecting
reproduction. Smaller body sizes are advantageous to diffusive processes and
therefore contribute to the improved hypoxia tolerance. The onset and duration
of hypoxic exposure during ontogenesis have a significant influence on Hb
oxygen affinity and body size. Transgenerational effects of hypoxia
acclimation could not be observed.
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Acknowledgments |
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