Metabolic plasticity and critical temperatures for aerobic scope in a eurythermal marine invertebrate (Littorina saxatilis, Gastropoda: Littorinidae) from different latitudes
Lab. Ecophysiology and Ecotoxicology, Alfred-Wegener-Institute for Polar and Marine Research, Columbusstr., 27568 Bremerhaven, Germany
* Author for correspondence at present address: Biology Dept, University of North Carolina at Charlotte, 9201 University City Blvd, Charlotte, NC 28223, USA (e-mail: isokolov{at}email.uncc.edu)
Accepted 26 September 2002
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Summary |
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Key words: temperature adaptation, aerobic scope, critical temperatures, respiration, metabolic cold compensation, Littorina
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Introduction |
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The study of the effects of environmental temperature on metabolic rates of
aquatic ectotherms has a very long history, and an enormous body of data on
this topic has been accumulated so far (reviewed by Clarke,
1980,
1998
). However, recent studies
have revealed that environmental temperature not only influences the total
metabolic rate in aquatic ectotherms but can also significantly affect
metabolic regulation, eliciting transition to anaerobiosis even in fully
oxygenated waters (reviewed by
Pörtner, 2001
;
Pörtner et al., 2001
).
Beyond the thermal optimum, whole-animal aerobic scope falls and eventually
vanishes at low or high critical temperatures (Tc I and
Tc II, respectively), when transition to anaerobic
mitochondrial metabolism occurs (Sommer et
al., 1997
; van Dijk et al.,
1999
; Frederich and
Pörtner, 2000
). Presumably, the temperature-induced
transition to partial anaerobiosis is caused by the progressively insufficient
capacity of circulation and ventilation and can provide only time-limited
survival of the animals at and beyond critical temperatures
(Tcs; Pörtner,
2001
). Comparisons between coldadapted Antarctic species and their
relatives (within a family or a class) from temperate waters have shown that
the Tcs in Antarctic ecthotherms are much lower than in
their temperate counterparts (Pörtner
et al., 1998
; Pörtner and
Zielinski, 1998
; van Dijk et
al., 1999
). This downward shift of the Tcs is
brought about by increased mitochondrial densities and increased thermal
sensitivity of mitochondrial oxygen demand, which may reduce the organism's
performance at high temperatures due to the high costs of mitochondrial
maintenance (Pörtner,
2001
). However, in Antarctic marine stenotherms, metabolic oxygen
demand is downregulated despite hugely elevated mitochondrial densities
(Pörtner et al., 1998
),
so that little or no metabolic cold adaptation of the metabolic rates is
observed (reviewed by Clarke,
1980
,
1998
;
Clarke and Johnston, 1999
).
Despite the importance of temperature-induced limitations of aerobic scope
for the metabolic physiology of animals, it is much less studied compared with
other aspects of the metabolic response to temperature. In particular, the
amount of data about the effects of latitudinal cold adaptation on the
temperature window of aerobic scope is very limited and practically absent for
eurythermal ectotherms. The only eurythermal species studied in this respect
is the intertidal polychaete Arenicola marina, which demonstrated
similar downward shifts of the Tcs as a result of
latitudinal cold adaptation and seasonal cold acclimatisation
(Sommer et al., 1997).
However, in the latter study, animals from different latitudes were acclimated
at their respective environmental temperatures (12°C and 6°C), thereby
confounding the effects of latitudinal adaptation and temperature acclimation
on Tcs. In order to yield a better understanding of the
broad applicability of the concept of Tcs, further studies
on organisms with different strategies of temperature adaptation are needed.
It is also very important to estimate the degree of phenotypic plasticity of
Tcs and the thermal window of aerobic scope in response to
temperature acclimation or acclimatisation, which are likely to be crucial for
eurythermal organisms that have to cope with considerable seasonal and
circadian temperature variation.
The intertidal Atlantic gastropod Littorina saxatilis is a
uniquely convenient object for the study of latitudinal cold adaptation. Its
distribution area covers more than 50° by latitude, ranging from North
Africa up to Svalbard (Reid,
1996). This allows us to avoid `apples with oranges' comparisons
of different species and to study warm- and coldadapted populations within an
ecologically, morphologically and genetically cohesive unit such as a single
species. Moreover, L. saxatilis, as with many intertidal species, is
at the eurythermal extreme of the eurythermstenotherm continuum and is
able to cope with wide and frequent temperature fluctuations typical for
intertidal habitats (Sokolova et al.,
2000a
). L. saxatilis can withstand temperatures of up to
35°C for at least several hours during summer low tides and up to 47°C
during short-term acute heating events
(Sokolova et al., 2000a
). This
species is also extremely cold- and freezetolerant. It can survive sub-zero
temperatures for several months and can tolerate temperatures as low as
-20°C for at least several hours
(Matveeva, 1974
;
Bourget, 1983
;
Sokolova and Berger,
2000
).
The aim of the present study was to analyse the effects of latitudinal cold adaptation on metabolic rate and critical temperatures in L. saxatilis from the temperate North Sea and sub-arctic White Sea. We measured aerobic metabolic rates in animals from different latitudes over a wide range of experimental temperatures and analysed the accumulation of end products of anaerobic metabolism at different temperatures. Concentrations of high-energy phosphates (ATP and phospho-L-arginine) were measured simultaneously in order to establish whether the transition to partial anaerobiosis was correlated with impaired intracellular energy status. In order to estimate the phenotypic plasticity of the metabolic response to the temperature change, rates of aerobic metabolism and critical temperatures were compared in snails from different latitudes acclimated to 4°C and 13°C. Analysis of the effects of latitudinal cold adaptation and temperature acclimation on metabolic rates and the temperature range of aerobic scope in L. saxatilis provides important insights into the metabolic responses to short-term temperature change vs long-term effects of acclimation or latitudinal adaptation in a eurythermal intertidal invertebrate. It also widens our perspective of the mechanisms of metabolic adaptation and regulation in response to temperature change in ectotherms. This study also complements our broader research concerning physiological, biochemical and populational mechanisms of adaptation of L. saxatilis to environmental stresses (temperature, salinity, oxygen deficiency) and addresses the basis of the extremely eurybiont life modus of this species.
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Materials and methods |
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In each study area, adult L. saxatilis (6-11 mm shell diameter)
were collected from small stones and gravel patches in the low intertidal
zone, within the brown macrophyte belt (Ascophyllum nodosum and
Fucus vesiculosus in the White Sea and F. vesiculosus and
Fucus serratus in the North Sea). Snails were transported alive to
the Alfred-Wegener-Institute (AWI) in Bremerhaven, Germany and acclimated in
aquaria with recirculated seawater set to the salinity of the respective
sampling sites (33.2-33.4 for the North Sea and 24.6-24.7
for
the White Sea) for at least 6-8 weeks prior to experimentation. In order to
reduce the effects of the circadian rhythm on the oxygen consumption of L.
saxatilis, snails were acclimated under conditions of constant dim light
and all experiments were started at the same time of day
(Sandison, 1966
;
Shirley and Findley, 1978
).
Prolonged laboratory acclimation was used to eliminate the potential
differences in metabolic physiology among the animals from populations, which
were due to their recent acclimatisation history in the field
(Hawkins, 1995
). This allowed
us to analyse irreversible (presumably, genetic) physiological differences
among the compared groups of snails. Two acclimation temperatures were used:
13°C, which was close to the respective field temperature at the time of
collection, and 4°C. Two aquaria were randomly assigned to each
combination of acclimation temperature and population of origin, and samples
for experimental incubations and measurements were withdrawn from them at
random. Water in the aquaria was continuously aerated, passed through a filter
and changed once every two weeks. Seawater from the Helgoland area was used,
and the required salinity was adjusted by adding artificial sea salt (Tropic
Marin, Wartenberg, Germany) or diluting with distilled water. Brown macroalgae
(F. vesiculosus) from Helgoland Island were added as a food source
ad libitum. No mortality was detected during transportation, and only
minimum mortality (<5%) was detected during laboratory acclimation.
Respiration rates
The routine rates of oxygen consumption in water were measured in closed
respiration chambers with Clarke-type electrodes connected to an oxygen
partial pressure monitor (PO2 monitor, Eschweiler, Kiel,
Germany). Two-point calibrations of temperature-equilibrated electrodes (in
air-saturated seawater for 100% readings and in
Na2SO3-saturated water for 0% readings) were performed
prior to and after each measurement.
Five to six animals of similar size [ranging from 40 mg to 50 mg (White Sea
populations) and from 80 mg to 120 mg wet tissue mass (North Sea populations)]
were used for each measurement. Prior to the experiments, the snails were
fasted for 72 h. Shells of the experimental snails were carefully scraped and
cleaned with 95% ethanol to remove potential microfouling. Snails were then
allowed to quickly recover in seawater, were blotted dry with tissue paper and
were put in the chamber filled with seawater at the respective acclimation
temperature (4°C or 13°C). Water in the chambers was continuously
mixed with magnetic stirring rods, which were placed under the nylon net
`floor' (mesh size, 1 mm) of the animal-containing main chamber. Respiration
chambers were placed in a temperature-controlled aquarium (total capacity,
751) maintained at the respective control temperature and allowed to
equilibrate for 30 min in order to minimize any effects of handling.
Preliminary experiments showed that 30 min habituation of experimental
chambers was sufficient to eliminate the overshoot effects of handling.
Temperature was raised by 1.5°C h-1 or lowered by 1°C
h-1. The rate of the temperature change was set by the capacity of
the water bath (HC F30, Julabo Labortechnik, Seelbach/Schwarzwald, Germany).
During this preliminary acclimation, free water exchange between the
experimental chambers and the surrounding water was allowed. After the
temperature reached the desired value, the respiration chambers were closed
and oxygen concentrations were monitored online for 15-25 min at the constant
temperature (±0.1°C) with continuous mixing. Decline in the oxygen
concentration was linear over this period, and repeated measurements of the
respiration rates of the same batch of animals at the respective control
temperatures indicated no change over at least 2 h. Oxygen tension was not
allowed to fall by more than 15% during each measurement. After each
measurement, blanks were run in the same chambers without animals in order to
account for the drift of electrodes and respiration by any microorganisms in
the experimental chamber and seawater. In order to minimize handling of the
experimental animals, they were placed in small cages made of plastic mesh
(mesh size, 1 mm) tightly fitted into the respiration chambers. During the
measurement of oxygen consumption, the cages were placed into the respiration
chambers, and during the blank reading they were carefully removed from the
chamber and placed on the bottom of the experimental aquarium. It should be
noted that the thermal limits and the rate of the temperature change used in
our experiments resembled the environmental values reported for temperate and
sub-arctic intertidal habitats (Sokolova
et al., 2000a). Water oxygen contents were calculated as µmol
l-1 O2 from oxygen PO2 readings
(measured in Torr) using temperature-dependent solubility coefficients for
oxygen. The determination of the chamber volume considered the volumes of
snails, stirring rods and nylon nets determined by fluid displacement. After
the experiments, animals were dissected in order to determine tissue wet mass
and the level of trematode infection. Respiration rates were expressed as
µmol O2 h-1 g-1 tissue wet mass. In the
determinations of the respiration rates, infested and uninfested animals were
pooled, as it has previously been demonstrated that infection by microphallids
(Trematoda) does not influence the respiration rate of Littorina spp.
(Lyzen et al., 1992
;
Sokolova, 1997
). There was
also no effect of the proportion of infested animals in a batch (which varied
from 0% to 60%) on the rates of oxygen consumption in the present experiment
(data not shown). Average trematode infestation rate across all samples was
24% (N=168) and 20% (N=129) in White Sea and North Sea
snails, respectively.
In this study, the temperature dependence of aerobic metabolism was
analysed by measuring the routine respiration rate in unfed L.
saxatilis. Measurements of the basal metabolic rate (BMR) in marine
gastropods is very difficult, because the animals tend to move continuously
unless anesthetized. We restricted the range of movements of the snails in our
experiments by providing them with minimum space so that the animals could
remain open and attached with their feet to the substratum but could not move
more than a few mm during the oxygen consumption rate
(O2) measurements. Earlier
studies had demonstrated that moderate crowding does not affect the metabolic
rates of Littorina (Vilenkin and
Vilenkina, 1973
). Thus, the routine metabolic rates we have
measured in our experiments are a good approximation of the resting metabolic
rates of L. saxatilis.
Temperature incubations and tissue collection
For the determination of the concentrations of high-energy phosphates and
anaerobic end products in the tissues of L. saxatilis at different
temperatures, samples of 400-500 animals were placed in large (101) mesh cages
of the same temperature-controlled experimental aquarium that had been used
for the measurements of the respiration rate. Incubations started at the
respective control temperatures (13°C or 4°C). Experimental animals
were allowed to equilibrate in the experimental aquarium overnight in order to
minimize the effect of handling, and then the temperature was raised by
1.5°C h-1 or lowered by 1°C h-1. The rate of the
temperature change was set by the capacity of the thermostat (HC F30, Julabo
Labortechnik, Seelbach/Schwarzwald, Germany). The duration of the longest
incubation was 22 h. During the preliminary acclimation and incubation
periods, the seawater in the aquarium was continuously aerated, mixed and
filtered by pumps. After each incubation, the water in the aquarium was
changed in order to prevent the accumulation of metabolic waste products.
After the temperature reached the desired value, a random sample of animals was taken out of the aquaria, animals were blotted dry, dissected and quickly inspected for trematode infestation. Infested specimens were discarded. In uninfested specimens, the foot muscle was quickly cut, blotted dry with tissue paper and frozen immediately in liquid nitrogen for subsequent metabolite analysis. For one sample, foot muscles of 8-12 specimens were pooled. At each experimental temperature, 5-9 samples were collected. Collection time was approximately 5 min per sample. During the collection of the tissues, temperature in the aquarium was maintained constant.
In order to test for the possible effects of experimental incubation per se on metabolite concentrations, control incubations were performed. Animals were kept for 26 h in the experimental aquaria at their respective control temperatures (4°C or 13°C), and tissues were sampled at the beginning and the end of this incubation period, as described above.
Metabolites
For the determination of metabolite concentrations using enzymatic tests,
samples of foot muscles were powdered with a pestle and mortar under liquid
nitrogen. Approximately 300 mg of tissue powder was homogenized in an excess
(5x) volume of precooled 0.6 mol l-1 perchloric acid (PCA)
with 10 mmol l-1 EDTA to bind tissue calcium, which proved
necessary for maximization of the measured ATP levels
(Sokolova et al., 2000b).
Precipitated protein was removed by centrifugation (2 min, 10 000
g). The extract was neutralized with 5 mmol l-1
potassium hydroxide to pH 7.0-7.5. Precipitated potassium perchloride was
removed by a second centrifugation. For ion chromatography, the extraction
procedure was identical except that EDTA was omitted. Preliminary experiments
showed that EDTA interfered with the determination of the concentrations of
short-chain organic acid by co-eluting with propionate. Extracts were stored
at -80°C.
For capillary electrophoresis, samples of foot muscles were powdered under liquid nitrogen. Approximately 100 mg of tissue powder was homogenized in 400 µl of ice-cold 15% trichloracetic acid with 0.1 g l-1 of tartrate used as internal standard. Samples were extracted for 5 min on ice, and precipitated protein was removed by centrifugation (2 min, 10 000 g). The supernatant was neutralized with 4 volumes of the mixture tri-n-octylamine: 1,1,2-trichlorotrifluoroethane (1:4). Organic phases were separated by centrifugation (3 min, 14 000 g), and the upper aqueous phase was collected, diluted 1:3 with ultrapurified water and directly applied to the capillary electrophoresis system (P/ACETM System MDQ, Beckman, Unterschleissheim, Germany) in order to determine concentrations of the short-chain organic acids (D- and L- lactate, malate, succinate and propionate). Separation was carried out in a 120 cm long eCAP capillary with a diameter of 75 µm (Beckman) at a constant temperature of 15°C in a buffer consisting of organic acid buffer (Agilent Technologies, Böblingen, Germany), acetonitrile and Brij 35 (20:5:1 v/v/v). The separation voltage of 27 kV was applied for 20 min to allow for the detection of the slowest moving component, propionate. Organic acids were detected by a photodiode array detector at 214 nm. The system was calibrated using standard mixtures of the respective organic acids subjected to the same extraction procedure as the tissue samples.
A reliable determination of acetate by capillary electrophoresis was not
possible owing to the presence of background acetate in samples extracted by
trichloracetic acid. Therefore, the concentration of acetate was measured in
PCA extracts according to a method modified from Hardewig et al.
(1991) using an ion-exclusion
column (IonPac ICE-AS 1, Dionex, Idstein, Germany) at a flow rate of 1 ml
min-1 and 40°C with 0.125 mmol l-1
heptafluorobutyric acid as an eluent. Peaks were monitored with a conductivity
detector. A micro membrane suppressor (AMMS-ICE, Dionex) was used to decrease
background conductivity. No acetate was detected in our samples.
Concentrations of ATP, L- and D-alanine were measured
spectrophotometrically using enzymatic tests (Bergmeyer, 1985). Concentrations
of phospho-L-arginine and L-arginine were assayed spectrophotometrically using
the enzymatic test described by Grieshaber et al.
(1978). Octopine dehydrogenase
for these determinations was purified from the adductor muscles of Pecten
maximus following the procedure described by Gäde and Carlsson
(1984
).
Chemicals
All chemicals were purchased from Sigma Chemical Co. (St Louis, MO, USA) or
Merck (Darmstadt, Germany). Enzymes were purchased from Roche Diagnostics
(Mannheim, Germany).
Derived indices and statistics
The relative amount of phosphagen (RPLA) in the total
phosphagen/aphosphagen pool was calculated according to the formula:
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Respiration rates (calculated as µg O2 consumed per hour per
g wet tissue mass) were standardized to the same wet tissue mass (50 mg)
according to equation 2:
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Statistical analysis was performed using mixed Model I analysis of variance
(ANOVA) after testing the assumptions of normal distribution and homogeneity
of variance of the data (Sokal and Rohlf,
1995). Factors `acclimation temperature' and `sea' were treated as
fixed, while factor `exposure temperature' was treated as random. There were
no differences between animals from the replicate aquaria maintained at the
same acclimation temperature in any of the studied parameters, so the data
were pooled for subsequent analysis. At the first step of an analysis,
three-way ANOVAs were performed estimating effects of factors `sea',
`acclimation temperature' and `exposure temperature'. If estimations of single
factor effects were impossible due to significant factor interactions, data
sets were split to perform two-way or one-way ANOVAs. Tukey's honest
significant deviance (HSD) test for unequal N was used as a method of
post-hoc comparisons, and the least-square method was employed for
planned comparisons (Sokal and Rohlf,
1995
).
The energy of activation (Ea) was determined from an
Arrhenius plot, i.e. log
O2 vs
1/T (K-1). The apparent Ea value was
calculated from the slope of the plot (i.e. slope =
Ea/2.303R) obtained by the least-square
linear regression (Sokal and Rohlf,
1995
). The Arrhenius breakpoint temperature (ABT) at which a
significant change in the slope (i.e. in Ea) occurs was
determined using an algorithm for fitting of two-segmented linear regressions
described by Yeager and Ultsch (1988). Slopes of regression lines were
compared according to Zar
(1996
). The highest
experimental temperature (32°C) characterized by the onset of heat coma
(see below) was excluded from the calculations of Ea and
ABT.
Differences were considered significant if the probability level of Type I error was <0.05. Results are expressed as percentages or mean values ± S.E. unless indicated otherwise.
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Results |
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Arrhenius breakpoint temperatures (ABTs), indicating discontinuity in the temperature dependence of aerobic metabolic rates, varied between 8°C and 18°C in different experimental groups. In North Sea snails, the ABTs were 8°C and 13°C in cold- and warm-acclimated animals, respectively. In White Sea snails, ABTs were 18°C and 10.5°C for cold- and warm-acclimated animals, respectively. The considerable difference in the ABTs between warm- and cold-acclimated groups from the White Sea was unexpected because the respiration rates did not differ significantly between these two groups at any experimental temperature (see above). This probably reflects a lower activation energy of aerobic respiration (Ea) in the range of 0-8°C in the cold-acclimated animals from the White Sea and a less sharp transition to the `temperature-independent' zone of metabolism (approximately 13-28°C; Fig. 1). The ABT calculated for the pooled data, including the warm- and cold-acclimated animals from the White Sea, was 10.5°C. In general, the increase of respiration rates with temperature was considerably slower beyond the ABT in all experimental groups from both locations. This was reflected in a significantly lower Ea at higher temperatures (P<0.05, d.f.=37-55): the Ea comprised 70-140kJ mol-1 in the temperature range between 0°C and the respective ABT and 10-35 kJ mol-1 beyond the ABT (Fig. 2). There was a trend for lower Ea values in White Sea animals as compared with their North Sea counterparts (Fig. 2).
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Critical temperatures: onset of anaerobiosis and changes of cellular
energy status
Exposure to high temperatures led to an onset of anaerobic metabolism in
L. saxatilis, as indicated by considerable succinate accumulation in
the foot muscle tissue (Fig.
3). Succinate was the only anaerobic end product accumulated in
L. saxatilis under these conditions. No lactate, acetate, propionate
or alanine accumulation was found. Incubation for 10 h or 26 h at the
respective control temperatures did not lead to changes in the concentrations
of succinate in White Sea or North Sea L. saxatilis
(t-tests, P>0.14 in all comparisons, N=4-6, data
not shown), suggesting that the temperature increase and not the incubation
per se was responsible for the switch to anaerobiosis. Thus, the
temperatures at which succinate accumulation significantly exceeded the
respective control levels were designated as the high critical temperatures
(Tc II). No low critical temperatures
(Tc I) were detected within the studied temperature range
in L. saxatilis.
Acclimation to low temperatures resulted in a considerable downward shift of the critical temperatures (Tcs) in North Sea L. saxatilis from 28°C in the warm-acclimated animals to 15°C in the cold-acclimated animals (Fig. 3A). In the White Sea animals, the Tcs changed only slightly with cold acclimation from 32°C to 28°C (Fig. 3B). It is worth noting that at and above the respective Tcs, the level of succinate accumulation was higher in White Sea animals compared with their North Sea counterparts. In addition to succinate accumulation, an increase in malate levels was observed at higher temperatures in L. saxatilis (Fig. 3C,D). By contrast, low temperatures (0-4°C) caused the malate concentrations to fall below the respective control levels in warm-acclimated animals from the two studied sites. In the cold-acclimated animals, the malate content of the foot muscle was similar in the control and at 0°C.
Incubation at high temperatures resulted in an impairment of cellular energy status, as visualized by the depletion of high-energy phosphates [the phosphagen phospho-L-arginine (PLA) and ATP] in Littorina. In general, North Sea animals tended to have higher control levels of PLA compared with their White Sea counterparts (3.3-3.1 µmol g-1 wet mass vs 2.7-2.8 µmol g-1 wet mass in North Sea and White Sea snails, respectively). However, these differences were only statistically significant in the cold-acclimated group (P=0.007). The concentrations of PLA were higher in the warm-acclimated animals from the White Sea compared with their cold-acclimated counterparts (F1,5=16.98, P=0.009). In North Sea animals, acclimation temperature did not significantly affect PLA concentrations (F1,5=2.93, P=0.15), although a similar trend is visible. The PLA concentration decreased significantly below the respective control level at or just below the critical temperatures (Fig. 4A,B). In parallel with the decrease of the PLA levels, the concentration of the respective aphosphagen (L-arginine) increased (Fig. 4C,D). The relative proportion of PLA (RPLA) in the total phosphagen/aphosphagen pool declined at or just below the Tcs, reflecting PLA depletion (Fig. 5A,B). Characteristically, the RPLA increased at low temperatures (0-4°C), probably due to the lower L-arginine concentrations in the animals incubated in the cold (Fig. 4C,D).
|
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ATP concentrations were similar in control animals from the White Sea and
the North Sea (P=0.10-0.50) and ranged between 1.7 µmol
g-1 wet mass and 2.2 µmol g-1 wet mass. Similar to
PLA, and as found in a previous study on fish (cf.
van Dijk et al., 1999), ATP
concentrations were significantly higher in warm-acclimated snails compared
with their cold-acclimated counterparts (F1,5=7.91,
P=0.04, and F1,5=273.7, P<0.001, for
North Sea and White Sea animals, respectively). Changes in intracellular ATP
concentration in L. saxatilis closely followed the depletion of the
phosphagen pool during temperature incubations
(Fig. 5C,D), but a significant
drop in ATP levels generally occurred later than the decrease of PLA
concentrations (cf. Figs 4,
5).
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Discussion |
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Acute temperature effects and the zone of `temperature independence'
of metabolism
The respiration rate of L. saxatilis rose quickly with increasing
temperatures until a point at or slightly above the respective acclimation
temperature was reached. Beyond this value, thermal sensitivity of
O2 was reduced
in a wide, 15°C range of experimental temperatures. This change in
metabolic regulation was also reflected in the ABTs of respiration rates
(8°C for cold-acclimated North Sea animals and 11-13°C for all other
groups). The Ea of aerobic metabolism was approximately
seven times lower above the ABT as compared with the Ea
below the ABT, reflecting diminished thermal dependence of aerobic metabolism.
It should be noted that both the control temperature (13°C) used in our
experiments as well as the ABTs were close to water temperatures at the time
of collection (September at the White Sea and October at the North Sea) and
were in the lower range of environmental temperatures experienced by the
snails in early autumn. Air and substrate temperatures during low tide could
be much higher (>20°C), especially on sunny days (I. M. Sokolova,
personal observation). Comparison with other gastropod species suggests that a
temperature insensitivity of aerobic metabolism at the high end of ambient
temperatures is typical for intertidal gastropods while not observed in their
subtidal counterparts. Thus, a temperate subtidal limpet Patella
oculus demonstrates a high temperature dependence of aerobic metabolism
over a wide range of environmental temperatures (10-25°C) with a
Q10 of 2.5-3.9, whereas in the two intertidal limpets
Patella granularis and Patella cochlear the metabolic rate
changes very little over the same environmental temperature range
(Q10=1.3-1.9) (Branch
et al., 1988
). In the intertidal tropical gastropods
Nodilittorina interrupta, Littoraria irrorata and Siphonaria
pectinata living at ambient water temperatures of 28-30°C, the
O2 was
practically temperature independent between 25°C and 40°C but changed
considerably with temperature in the subtidal snail Stramonita
haemastoma from the same area
(McMahon, 1992
).
Interestingly, even among Antarctic species, the intertidal gastropod
Nacella concinna, which experiences fluctuating temperatures during
daytime, had a temperature-independent metabolic rate over a temperature range
close to its environmental range (-1.7°C to +0.5°C), in contrast to
the subtidal species Pelilittorina setosa and Trophon sp.,
which showed a significant increase in
O2 with
increasing temperature (Houlihan and
Allan, 1982
). Possibly, the relative thermal independence of
metabolic rate at high temperatures can be adaptive for intertidal animals,
allowing them to minimize energy expenditure during fast and frequent
temperature fluctuations at low tide.
In molluscs, high thermal sensitivities of metabolism within the
environmental range were associated with increased longterm metabolic costs
and with a lower tolerance to extreme temperatures
(Hawkins, 1995), suggesting
that a reduced temperature dependence of metabolic rate can be selectively
advantageous in thermally unstable intertidal environments. Thermal
insensitivity of metabolism may also reflect progressive metabolic depression
at high temperatures in L. saxatilis. Whichever is the case, such an
`energy conservation' strategy can be especially important for high shore
species such as L. saxatilis that are resource limited due to the
short feeding periods that are generally restricted to a short period around
high tide when the substrate is wet. By contrast, subtidal species are not
food limited and experience smaller temperature fluctuations than their
intertidal counterparts. They adopt an `exploitative strategy' that allows
them to utilize resources at a high rate in order to maximize growth, and
their metabolic rates increase with increasing temperature
(Branch et al., 1988
). The
physiological mechanisms allowing intertidal eurytherms to maintain a
temperature-independent rate of metabolism in a wide range of environmental
temperatures are not known and would be an interesting subject for further
studies.
Heat-induced loss of aerobic scope
Acute temperature increase resulted in a considerable metabolic disturbance
in L. saxatilis at critically high temperatures indicated by the
onset of anaerobic metabolism and the adverse changes in the cellular energy
status. In warm-acclimated L. saxatilis from the White Sea and the
North Sea, the onset of anaerobiosis between 28°C and 32°C coincided
with entering heat coma, characterized by the loss of nervous integration and
muscle relaxation (Clarke et al.,
2000a). The onset of heat coma in gastropods can be easily
determined by the loss of contractility of the foot muscle and the
characteristic inward curling of the lateral foot area (Clarke et al.,
2000a
,b
).
In cold acclimated L. saxatilis, heat coma was also observed at
32°C, although the onset of anaerobiosis occurred at lower temperatures
(18°C and 28°C in North Sea and White Sea snails, respectively). The
heat coma temperature (HCT) in our experiments was within the range of the
mean HCTs reported for L. saxatilis from some North Atlantic
populations (30.8-31.7°C) (Clarke et
al., 2000b
).
It has been suggested that neuromuscular failure in ectotherms may become
detrimental later than oxygen limitation due to more extreme denaturation
temperatures of proteins in general, including those involved in neuromuscular
functions (Pörtner, 2001;
Sokolova and Pörtner,
2001b
). On the other hand, extreme hypoxia may also contribute to
limiting neural function, in line with a suggested systemic to molecular
hierarchy of thermal limits (Pörtner,
2002
). Our data suggest that in an extremely eurythermal species
such as L. saxatilis, the neuromuscular failure indicated by the
onset of heat coma and oxygen limitation may go hand in hand, at least in
warm-acclimated animals with wide temperature windows of aerobic scope. Unlike
most vertebrate ectotherms, the neuromuscular failure associated with heat
coma is a fully reversible condition in gastropods (Clarke et al.,
2000a
,b
;
I. M. Sokolova, personal observations), and entry into heat coma generally
occurs at temperatures well below acute lethal limits (45-47°C in L.
saxatilis; Sokolova et al.,
2000a
). This may reflect the lower level of integration in
invertebrates and relatively smaller role of the central nervous system in
survival of acute environmental stress in these organisms.
Heat coma and the associated neuromuscular failure at extremely high
temperature (32°C) are likely to contribute to capacity limitations and,
finally, disintegration of oxygen uptake and/or delivery systems. In marine
molluscs, the ventilation of the mantle cavity is maintained by ciliary
activity, which may be reduced upon entry into heat coma and may decrease
oxygen delivery (Sandison,
1967). It is not unreasonable to assume that the loss of neural
integration would also interact with a loss in cardiac function, although
direct evidence for this is absent for marine gastropods. In general, the
contention that neuromuscular failure is involved in the development of oxygen
limitation and associated metabolic disturbances would not be all that
surprising. However, our data suggest that the heat coma per se is
not a primary mechanism of oxygen limitation in Littorina. First of
all, in cold acclimated L. saxatilis, the onset of anaerobiosis
occurred at temperatures well below HCT (31-32°C), implying that other
physiological limitations are involved. Secondly, the depletion of the
phosphagen PLA was observed below the HCT in both warm- and cold-acclimated
snails, indicating progressive discrepancy between energy demand and supply
that is not related to neuromuscular failure. A decline in PLA levels was
closely paralleled by the onset of anaerobiosis, suggesting that
temperature-induced anaerobiosis was linked to insufficient energy production
to cover high energy demands at elevated temperatures. In support of these
considerations, there was a significant negative correlation between
respiration rate and succinate accumulation at 32°C in L.
saxatilis (r=-0.971, N=4, P=0.029), supporting
the notion that anaerobiosis is involved in the compensation for insufficient
aerobic energy production at elevated temperatures.
It is interesting to note that at extremely high temperatures (32°C),
White Sea L. saxatilis had significantly lower respiration rates
associated with considerably higher levels of succinate accumulation as
compared with their North Sea counterparts. This contrasts with findings in
the polychaete A. marina, where both higher rates of oxygen turnover
and higher levels of succinate were found in White Sea compared with North Sea
specimens at extreme temperatures (Sommer
and Pörtner, 2002). High levels of succinate accumulation
were previously reported for three Littorina spp.
(Sokolova et al., 2000b
), the
blue mussel Mytilus edulis
(Sukhotin and Pörtner,
1999
) and A. marina
(Sommer et al., 1997
) from the
White Sea as compared with their temperate counterparts, indicating a higher
reliance on mitochondrial anaerobic metabolism at critical temperatures in the
sub-arctic White Sea animals. Nonetheless, anaerobic metabolism was not
sufficient to keep up with the energy demand at high temperatures, as
indicated by the progressive decline in ATP levels.
Temperature-induced mitochondrial anaerobiosis in L. saxatilis is
probably brought about by insufficient oxygen supply to tissues before
disruption of mitochondrial function occurs at critically high temperatures.
At first sight, dysfunction of the electron transport systems and/or
denaturation of key aerobic mitochondrial enzymes at extremely high
temperatures might elicit the transition to anaerobiosis and the formation of
anaerobic end products even in mitochondria that are not oxygen limited.
However, in fish and bivalves, the respiration rate of isolated mitochondria
was not impaired over a broad temperature range that was well beyond the
critical temperatures and even lethal temperature limits, suggesting that
aerobic scope at critical temperatures is not limited by the thermal
intolerance of mitochondrial function
(Weinstein and Somero, 1998;
Pörtner et al., 1999
on
Laternula elliptica gill mitochondria;
Hardewig et al., 1999
;
van Dijk et al., 1999
). We do
not have data on the heat stability of isolated mitochondria in L.
saxatilis. However, denaturation temperatures of key mitochondrial
enzymes in this species are far beyond the critical temperatures of aerobic
scope and range between 40°C and 45°C
(Sokolova and Pörtner,
2001b
). This agrees with the findings of high thermal tolerance of
mitochondrial enzymes and isolated mitochondria in other species as compared
with the whole-organism thermal limits
(Weinstein and Somero, 1998
;
Pörtner et al., 1999
;
Hardewig et al., 1999
;
van Dijk et al., 1999
).
Interestingly, indirect evidence that the heat-induced anaerobiosis in
L. saxatilis is due to insufficient oxygen supply to tissues is also
provided by the comparison of aerobic and anaerobic metabolic rates in water
vs air in this amphibious species. This specialized high-shore
gastropod possesses a vascularized area of mantle cavity that can serve as a
lung in addition to a ctenidium and is equally well adapted to breathe
atmospheric and water-born oxygen (Fretter
and Graham, 1976; McMahon,
1988
). However, ambient oxygen levels in air are approximately 30
times higher than in seawater, implying higher oxygen availability and lower
costs of ventilation in air-breathing animals. During prolonged air exposure
at 30°C, more than 98% of total (aerobic and anaerobic) ATP turnover in
L. saxatilis was supplied by aerobic pathways
(Sokolova and Pörtner,
2001a
). Moreover, succinate accumulation, which indicates
mitochondrial anaerobiosis, was very low, and the main anaerobic end product
in air was alanine (Sokolova and
Pörtner, 2001a
), suggesting that mitochondria remained
predominantly aerobic, and anaerobic metabolism was mostly restricted to the
cytosolic compartment (Grieshaber et al.,
1994
). Succinate accumulation contributed only 5-12% to anaerobic
ATP turnover during air exposure at high temperatures in L.
saxatilis, the rest being supplied by alanine accumulation and depletion
of high-energy phosphates. By contrast, exposure of L. saxatilis to
similar temperatures in water led to enhanced levels of succinate
accumulation, indicating more severe mitochondrial anaerobiosis. Anaerobic
metabolism contributed approximately 2.5% to total (aerobic and anaerobic) ATP
turnover in 4°C-acclimated North Sea animals and 16-43% in other
experimental groups (Fig. 6). A
low anaerobic contribution in cold-acclimated North Sea animals reflects
exceptionally high aerobic metabolic rates at high temperatures in this
experimental group. Succinate accumulation accounted for 76-97% of total
anaerobic ATP turnover rate
(
ATP) during
temperature-induced anaerobiosis of L. saxatilis in water
(Fig. 7). This comparison
suggests that the temperature-induced limitation of aerobic scope in L.
saxatilis is alleviated in air, probably due to the higher oxygen
availability in this milieu. This also supports our proposition that the
transition to partial mitochondrial anaerobiosis at critically high
temperatures in L. saxatilis is caused by insufficient oxygen uptake
and/or delivery rather than by the heat-induced disruption of mitochondrial or
neural function. It is worth noting that, in order to test this hypothesis and
further elucidate the role of variable oxygen concentration in heat-induced
anaerobiosis of this species, additional studies involving experimental
manipulations of oxygen content in the medium are required.
|
|
Effects of latitudinal adaptation vs cold acclimation on
metabolism
The rate of aerobic metabolism was similar in White Sea and North Sea
animals acclimated at the control temperature (13°C), indicating no
metabolic cold adaptation in respiration rates in sub-arctic L.
saxatilis. These results contrast with our previous findings that the
activity of several key metabolic enzymes is higher in White Sea L.
saxatilis than in their North Sea counterparts
(Sokolova and Pörtner,
2001b). Interestingly, similar results were reported for pectinid
bivalves, where a considerably higher activity of a key aerobic enzyme,
citrate synthase, in a cold-adapted Antarctic scallop (Adamussium
colbecki) compared with its temperate counterpart (Aequipecten
opercularis) did not result in a compensation of the aerobic metabolic
rate at the whole organism level
(Heilmayer et al., 2002
). In
the roach Rutilus rutilus, seasonal cold acclimatisation and cold
acclimation led to a compensatory increase in the activity of several key
metabolic enzymes and of Na+/K+-ATPase but did not
result in the overall increase of the metabolic rate
(Koch et al., 1992
). This
shows that increased enzymatic capacity is not always directly translated into
increased metabolic flux and emphasizes the complex relationships between
enzyme capacity and the regulation of enzyme in the whole-animal.
Metabolic responses to cold acclimation differed in L. saxatilis
from the North and the White Seas. In the temperate North Sea animals, cold
acclimation led to an increase in aerobic metabolic rates and the concordant
downward shift of the critical temperature by nearly 15°C. It should be
noted that an increase in the respiration rate as a result of cold acclimation
is a well-documented phenomenon in aquatic ectotherms and matches an increase
of mitochondrial volume density and/or capacity
(Egginton and Sidell, 1989;
Guderley, 1998
;
St-Pierre et al., 1998
). At
least in eurytherms, increased mitochondrial density leads to a rise in the
baseline energy demand due to the high costs of mitochondrial maintenance that
are no longer met by oxygen uptake at higher temperatures, and the higher the
density of mitochondria (other things being equal), the lower is the critical
temperature when oxygen uptake and/or delivery becomes insufficient and onset
of anaerobiosis is observed (Pörtner,
2001
). It is plausible to suggest that the simultaneous increase
in the
O2 and
the downward shift in the Tc in eurythermal North Sea
L. saxatilis may have a similar physiological background. A downward
shift of Tc associated with increased mitochondrial
densities has been previously shown in the cold-adapted lugworms A.
marina (Sommer et al.,
1997
; Sommer and Pörtner,
2002
). By contrast, cold acclimation in the White Sea L.
saxatilis did not significantly affect
O2 or
Tcs of aerobic scope, suggesting that cold acclimation may
have had no effect on mitochondrial oxygen demand in these animals.
As a corollary, Tcs characterized by the onset of
anaerobiosis proved to be very plastic and responsive to cold acclimation in
North Sea L. saxatilis but not in their White Sea counterparts. In
White Sea snails, the temperature window of aerobic scope was very wide and
remained practically unchanged by cold acclimation. There was also no effect
of cold adaptation or acclimation on aerobic metabolic rates in sub-arctic
L. saxatilis compared with their temperate counterparts. These
evidently opposing strategies may be related to the differences in winter
environmental conditions between the two study sites. At the North Sea site,
the winter temperatures are approximately 3-6°C, and intertidal animals
usually remain active, feed and grow throughout the winter
(Hickel et al., 1997;
Janke, 1997
; I. M. Sokolova,
personal observation). A compensatory increase in metabolic rate due to cold
acclimatisation in these animals could help them to maintain relatively high
activity levels at low temperatures. By contrast, in the White Sea, the winter
is associated with extremely low temperatures (down to -1.5°C) and greatly
diminished food availability due to light-limited algal growth in this
sub-arctic area (Matveeva,
1974
; Babkov,
1998
). White Sea Littorina spend the winter in an
inactive state in low intertidal and subtidal horizons, where they migrate
during late autumn (Matveeva,
1974
; Galaktionov,
1993
; I. M. Sokolova, personal observations). Accordingly, they do
not demonstrate a compensatory increase in respiration during cold acclimation
(this study). A depression of metabolic rates, which correlates with reduced
levels of feeding and general activity, was observed in many marine and
freshwater molluscs in winter (Innes and
Houlihan, 1985
) and is consistent with an important role of
hypometabolism as a survival strategy during prolonged periods of cold
exposure or resource limitation. This strategy is found across a wide variety
of phyla, from resting bacterial spores through estivating and dormant
invertebrates to hibernating mammals
(Hochachka and Guppy, 1987
).
In general, the intriguing differences in the metabolic response to
acclimation between L. saxatilis from different latitudes call for
further investigation in order to establish the different effects of cold
acclimation on metabolic rates at the cellular and sub-cellular level, e.g.
with respect to mitochondrial densities and capacities. These findings also
warrant further investigations on populations from a wide range of latitudes
and thermal habitats in order to allow generalizations about the effects of
latitude on critical temperatures of aerobic metabolism and metabolic
plasticity in eurythermal invertebrates.
In conclusion, our study demonstrates that temperature-induced oxygen
limitation also occurs in an extremely eurythermal intertidal invertebrate,
suggesting that mitochondrial hypoxia and anaerobiosis at high temperatures
are a common feature of ectotherm physiology (cf. Pörtner,
2001,
2002
). The heat-induced onset
of anaerobiosis is presumably due to the limited capacity of oxygen uptake and
transport mechanisms rather than a result of neural and/or mitochondrial
dysfunction and can (partly) be alleviated in the presence of high ambient
concentrations of oxygen, e.g. in air. The heat-induced metabolic disturbances
lead to a progressive discrepancy between energy demand and energy supply and
result in a time-limited situation, which can prove lethal if the adverse
temperature conditions persist. It should be noted that extreme temperatures
ranging between 35°C and 45°C are not uncommon in the natural habitats
of L. saxatilis in summer even in the sub-arctic White Sea area
(Sokolova et al., 2000a
),
suggesting that the metabolic machinery may function close to its
physiological limits in this extremely eurythermal species. This also implies
that L. saxatilis populations may become vulnerable to the global
temperature change, especially in such ecologically marginal habitats as high
shore levels characterized by extreme temperature fluctuations (Sokolova et
al.,
2000a
,b
).
Notably, Stillman and Somero
(2000
) arrived at a similar
conclusion in their study of upper thermal limits of 20 species of crabs of
genus Petrolisthes, which suggested that the upper thermal tolerance
limits of some intertidal species may be near current habitat temperature
maxima, and global warming therefore may affect the distribution limits of
intertidal species to a greater extent than their subtidal counterparts
(Stillman and Somero, 2000
).
Differences in the metabolic response to temperature acclimation in White Sea
and North Sea Littorina shown in this study require further
investigation and emphasize the importance of taking into account other
relevant factors (e.g. seasonal amplitude of temperatures, food availability,
activity levels of animals) when studying metabolic adaptations to
temperature.
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Acknowledgments |
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