Size- and age-dependent changes in adductor muscle swimming physiology of the scallop Aequipecten opercularis.

2492
INTRODUCTION
Ageing is a multifactorial process which involves progressive
deterioration of cells, tissues and organs that altogether causes a
decline in physiological functioning in the whole organism over their
life time. A major paradigm of cellular aging holds the life long
production of reactive oxygen species (ROS) in aerobic metabolism
responsible for damaging cell constituents such as lipids, proteins and
DNA and altering the function of these biomolecules. We have been
investigating ROS production and turnover in bivalve molluscs for
several years now and can track the progressive accumulation of ROS-
mediated damage and decline of cellular function with age in different
bivalve species (Philipp et al., 2005a; Philipp et al., 2006; Philipp et
al., 2005b; Strahl et al., 2007). This led us to conjecture that the
constraints in cellular and metabolic functioning posed by the aging
process may also impair the physiological response capacity towards
environmental stress in aged individuals at the whole organism level.
Bivalves prove to be attractive models to study ageing for several
reasons. They are ectotherms, they thrive in considerably diverse
habitats and have thus evolved different life styles, aside from their
shells providing a readable record of their chronological age. Thus,
in contrast to other classical ageing models such as rats, mice, D.
melanogaster and C. elegans, it is possible to compare bivalves from
various environments and determine their individual chronological
age and natural maximum life span in each local population. This
information can be further used to establish and combine the influence
of intrinsic (genetic) and extrinsic/environmental (e.g. predation
pressure) factors on the ageing process.
Mitochondria are centrally involved in the process of cellular
aging and extremely vulnerable to ROS damage. Their membranes
contain a high degree of unsaturated fatty acids and the
mitochondrial DNA lies, unprotected by histones, in close vicinity
to the ROS generating respiratory chain systems of the inner
mitochondrial membrane.
Metabolic rate can influence ROS generation rate (for reviews,
see Jackson, 2005; Sen and Packer, 2000), thus, an exercise-induced
increase of metabolic oxygen turnover may possibly be connected
to increased ROS generation, leading to mitochondrial damage.
However, during moderate exercise, ROS generation rates may even
become reduced as mitochondria enter state three (substrate and
ADP available), where the lowest possible membrane potential and
the lowest ROS generation rate have been found to occur (Philipp
et al., 2005b). Accordingly, the changes in ROS formation obtained
by using exercise studies are diverse (Jackson, 2005). Studies on
mammals for example, using different levels of treadmill running
(exhaustive, moderate, low) resulted in increased, no, or even
decreased lipid peroxidation (malondialdehyde accumulation)
(Lovlin et al., 1987).
In the present study we investigated the effects of exercise
physiology on physiological ageing in the scallop Aequipecten
opercularis. The queen scallop Aequipecten opercularis from the
Isle of Man, UK is short lived [8 to 10 years (Ansell et al., 1991;
Philipp et al., 2006)] and belongs to an active swimming ecomorph
within the scallop group (Minchin, 2003). These animals actively
swim and change their location to avoid unfavourable
environmental conditions including the escape from predators
(Paul, 1980; Wong and Barbeau, 2003). A decline in physiological
fitness, e.g. exercise capacity with age, has been reported for
humans, rat and mice and has been related to a decline in
The Journal of Experimental Biology 211, 2492-2501
Published by The Company of Biologists 2008
doi:10.1242/jeb.015966
Size- and age-dependent changes in adductor muscle swimming physiology of the
scallop Aequipecten opercularis
Eva E. R. Philipp1,*, Maike Schmidt2, Carina Gsottbauer3, Alexandra M. Sänger3 and Doris Abele1
1Alfred-Wegener-Institute for Polar and Marine Research, Department of Biosciences, 27570 Bremerhaven, Germany,
2Center of Biomolecular Interactions Bremen, University of Bremen Faculty 2 (Biology/ Chemistry), D-28334 Bremen, Germany and
3Department of Organismic Biology, Zoology and Functional Anatomy, Vascular and Muscle Research, University Salzburg, Austria
*Author for correspondence (e-mail: eva.philipp@awi.de)
Accepted 15 May 2008
SUMMARY
The decline of cellular and especially mitochondrial functions with age is, among other causes, held responsible for a decrease
in physiological fitness and exercise capacity during lifetime. We investigated size- and age-related changes in the physiology of
exercising specimens of the short lived swimming scallop Aequipecten opercularis (maximum life span 8 to 10years) from the Isle
of Man, UK. A. opercularis swim mainly to avoid predators, and a decrease in swimming abilities would increase the risk of
capture and lower the rates of survival. Bigger (older) individuals were found to have lower mitochondrial volume density and
aerobic capacities (citrate synthase activity and adenylates) as well as less anaerobic capacity deduced from the amount of
glycogen stored in muscle tissue. Changes in redox potential, tissue pH and the loss of glutathione in the swimming muscle
during the exercise were more pronounced in young compared to older individuals. This indicates that older individuals can more
effectively stabilize cellular homeostasis during repeated exercise than younger animals but with a possible fitness cost as the
change in physiology with age and size might result in a changed escape response behaviour towards predators.
Supplementary material available online at http://jeb.biologists.org/cgi/content/full/211/15/2492/DC1
Key words: ageing, bivalves, exercise, ROS.
THE JOURNAL OF EXPERIMENTAL BIOLOGY

2493Swimming physiology of aged scallops
mitochondrial function and mitochondrial volume density with
age, as there is a positive correlation between mitochondrial
function and volume density to fatigue resistance and exercise
capacity (Creed et al., 2004). Decreasing swimming capacity with
age could lead to a higher risk of capture and lower survival rate
of aged individuals.
In a previous study investigating clap rates and swimming, as
well as valve closure behaviour, we examined the swimming
capacity in smaller and bigger A. opercularis individuals. We
observed no difference in clap rate, but found that nearly 50% of
young scallops closed their shells completely and for as long as
30 min following the predator attack. By contrast, just 3% of the
bigger individuals behaved in this way and the majority remained
permanently open or opened the shells again within the 30 min
experimental observation (Schmidt et al., 2008).
Based on these behaviour experiments, we assumed a change of
cellular physiology to occur in A. opercularis with size and age,
which might involve a decrease in anaerobic and aerobic energy
generating capacities, caused by a decline of mitochondrial function
and/or volume density with size and age. Therefore, in the present
study we investigated components of the aerobic and anaerobic
energy generating systems, as well as redox potential, antioxidant
defence systems and markers of oxidative damage during exercise
in the phasic (striated) adductor muscle of smaller (younger) and
bigger (older) individuals, to see whether the observed change in
swimming behaviour with size is at least partly due to physiological
changes.
MATERIALS AND METHODS
Sampling and maintenance
Irish Sea queen scallops, Aequipecten opercularis Linnaeus 1758,
were dredged 12miles south of Port St Mary, Isle of Man in July
2005 at about 51m water depth. Animals were transported to the
Port Erin Marine Laboratory and kept in natural sea water flow-
through aquaria at ~14°C and 34 PSU for several days. Animals
were transported in thermoboxes with wet cotton wool and
supplemented with oxygen and cold packs to the Alfred-Wegener-
Institute of Polar and Marine Research, Germany. A. opercularis
individuals were kept in flow-through aquaria at ~10°C (mean in
situ temperature) and 34PSU for 2weeks prior to experimentation.
Animals were fed twice a week with life plankton (DT’s Live Marine
Phytoplankton®, Sycamore, IL, USA).
Individuals were be grouped in two classes: small animals from
40–55mm (below marketable size) (Jenkins et al., 2003) and bigger
animals from 65–75mm shell height. Age was determined with the
von Bertalanffy growth function (VBGF) of Philipp et al. (Philipp
et al., 2006) obtained for A. opercularis from the same sampling
station. The mean age of the small animals was 2±0.5years (mean
± s.e.m.) and for the big animals it was 4±0.5years.
Swimming experiments
Experimental design
Scallops were fixed with a Teflon screw within the experimental
setup and the whole system was video recorded as described by
Schmidt et al. (Schmidt et al., 2008) (see Supplementary material
Movie 1).
Swimming was triggered by the sea star Asterias rubens, a natural
predator in the environment of A. opercularis. The swimming
experiment (Table1) consisted of a 30min acclimation phase to
experimental conditions (unstressed animals, group 0), run 1: 1min
predator stress (group1) and 15min recovery (group 2), run 2: again
1min predator stress (group3) and 15min recovery (group4).
During every 1min swimming period A. opercularis individuals
were touched up to ten times by the sea star even if the shell was
closed. Directly after the respective event, individuals of each group
were removed from the experimental aquaria quickly dissected and
the adductor muscle snap frozen in liquid nitrogen for biochemical
analysis.
Isolation of adductor muscle mitochondria
For identification of general function of adductor muscle
mitochondria of unstressed A. opercularis, state 2 and 3 respiration
with succinate or glutamate as energetic substrates, and H2O2
generation in state 2 were investigated in 16 mitochondrial isolates.
Mitochondria were isolated from the muscle tissue of freshly
sacrificed bivalves, ranging in size from 51.5–70mm shell height,
using the method of Guderley et al. (Guderley et al., 1995).
Tissues of one or two A. opercularis specimens were pooled for
one experiment. About 3 g of muscle tissue were finely chopped
in four to five times the volume of homogenisation buffer
(480 mmol l–1 sucrose, 30 mmol l–1 Hepes, 230 mmol l–1 KCl,
3 mmol l–1 Na2-EDTA, 6 mmol l–1 EGTA, 5 mmol l–1 MgCl2, 1%
bovine serum albumin, 1μl ml–1 aprotinine, pH 7.0). Briefly, the
tissue was homogenised in a pre-cooled glass and Teflon
homogeniser, centrifuged at 900g for 10 min at 2°C and the
supernatant collected. The supernatant was centrifuged at 10 000g
for 10 min to sediment the mitochondria. The resulting pellet was
resuspended in 1 volume of isolation medium without MgCl2 and
centrifuged at 10 000g for 10 min. The final pellet was resuspended
in magnesium-free isolation medium containing 1% BSA
(1 g 100 ml–1) and 1μg ml–1 aprotinine.
Respiration of isolated mitochondria
Mitochondrial respiration was measured at 10°C in respiration
buffer (480mmoll–1 sucrose, 70mmoll–1 Hepes, 158mmoll–1 KCl,
10mmoll–1 KH2PO4, 50mmoll–1 taurine, 50mmoll–1 β-alanine,
pH7.4). The substrate used was 24mmoll–1 succinate, with 5μmoll–1
rotenone to prevent respiration of endogenous NAD-linked substrates
(Brand, 1995), or 29mmoll–1 glutamate, and state 3 respiration was
induced by addition of 0.6mmoll–1 ADP. Respiration rate was
recorded with oxygen microoptodes® (PreSens GmbH, Neuweiler,
Germany). Oxygen concentrations were calculated using the oxygen
solubility (βO2) according to Johnston et al. (Johnston et al., 1994)
and the atmospheric pressure of the day.
Production of hydrogen peroxide (H2O2) by isolated mitochondria
Mitochondrial hydrogen peroxide production was measured
fluorimetrically by recording the reaction of H2O2 with homovanilic
Table1. Group allocation in the swimming experiment
Run 1 Run 2
30 min acclimation Sea star attacks 1 min 15 min recovery Sea star attacks 1 min 15 min recovery
= group 0 = group 1 = group 2 = group 3 = group 4
THE JOURNAL OF EXPERIMENTAL BIOLOGY

2494
acid (HVA; λexitation=312nm and λemission=420nm) in the presence
of horseradish peroxidase (HRP), using a procedure modified after
Miwa et al. (Miwa et al., 2003). The H2O2 generation rate of A.
opercularis mitochondria was measured with a LS 50B Perkin Elmer
fluorometer with a cooled sample compartment and magnetic stirring.
The H2O2 generation in state 2 was recorded for each mitochondrial
aliquot directly in the fluorometer. A subsample of 150μl A.
opercularis mitochondrial solution was incubated with 850μl assay
medium to which 0.1mmoll–1 HVA and 2.5 i.u.ml–1 HRP at 10°C
were added. As soon as the fluorescence signal steadied, the
following chemicals were added in the order: (1) 24mmol l–1
succinate, 5μmoll–1 rotenone, 100i.u.superoxide dismutase (SOD),
20μmol l–1 antimycin; (2) 29mmol l–1 glutamate, 100 i.uSOD,
20μmol l–1 antimycin. Alternatively, in the order glutamate,
antimycin, SOD, where rotenone is an inhibitor of respiratory
complex I and antimycin an inhibitor of respiratory complex III.
In each experiment, fluorescence was calibrated with an H2O2
standard (0.2nmol l–1; Merck, Darmstadt, Germany). Both, H2O2
generation rates and oxygen consumption rates were measured in
parallel and related to mitochondrial protein content (see Keller et
al., 2004).
Mitochondrial density
For assessment of mitochondrial density a total of 14 (seven young
and seven old) samples of the striated adductor muscle were
dissected, immersion-fixed in Karnovsky’s (Karnovsky, 1965)
paraform–aldehyde–glutaraldehyde fixative, post-fixed in 1%
osmium tetroxide, dehydrated in a graded series of ethanols and
embedded in resin.
Ultrathin sections were cut on a Reichert-Jung (Vienna, Austria)
Ultracut microtome and mounted on Formvar-coated 75- and 100-
mesh copper grids. The sections were then contrasted with aqueous
solutions of uranyl acetate and lead citrate and viewed in a Zeiss
EM 910 transmission electron microscope (see Fig. 1, TEM). Based
on randomised photosampling, 40 micrographs per muscle tissue
sample were taken and the volume densities of mitochondria per
muscle fibre VV(Mito/Mf) were determined using stereological
methods (Weibel, 1979).
After testing, normality and equal variances differences between
the two age groups were assessed with a two-sided unpaired t-test
(Sigma Stat 3.1).
Biochemical measurements
Citrate synthase (EC 4.1.3.7)
Frozen muscle tissue of control animals was ground in liquid
nitrogen and homogenised with a glass homogeniser (Nalgene,
Rochester, NY, USA) in Tris–HCl buffer [20mmol l–1 Tris–HCl,
1 mmol l–1 EDTA, 0.1% (v/v) Tween 20, pH 7.4] 1:10 (w/v).
Homogenates for citrate synthase (CS) activity were sonicated for
15min in a Branson Sonifier 450 (output control 8, duty cycle 50%)
cooled to 0°C and centrifuged at 7400g for 5min at 2°C. CS activity
was measured after Sidell et al. (Sidell et al., 1987) recording the
absorbance increase of 0.25 mmol l–1 DTNB [5,5�-dithiobis(2-
nitrobenzoic acid)] in 75mmol l–1 Tris–HCl (pH8.0), 0.4mmol l–1
acetyl-CoA and 0.4mmol l–1 oxaloacetate at 412nm. Activity was
calculated using the millimolar extinction coefficient ε412 mmol l–1
of 13.61mmol l–1 cm–1.
ATP, ADP and AMP
Adenylate concentrations were measured after Lazzarino et al.
(Lazzarino et al., 2003) using high performance liquid
chromatography (HPLC).
E. E. R. Philipp and others
Frozen muscle tissue was ground in liquid nitrogen and
homogenised with a micropistill in a 1.5ml reaction vial with ice-
cold, nitrogen saturated precipitation solution [CH3CN (acetonitrile)
+ 10mmoll–1 KH2PO4, at a ratio of 3:1, pH7.4] at a 1:10 (w:v) tissue
to medium ratio. Precipitation solution was prepared weekly and the
pH checked immediately before use. The homogenate was
centrifuged at 20690g for 10min at 4°C and the clear supernatants
were stored on ice. Pellets were supplemented with 1ml of the
precipitation solution and resuspended for several seconds using an
ultraturrax, centrifuged again as above, and the supernatants
combined. This extract was washed with the double volume of
chloroform (10sec vortexing with HPLC grade CH3Cl) and
centrifuged as above. The upper aqueous phase, containing the water-
soluble low molecular mass compounds, was collected and washed
again twice with chloroform. Supernatants were then stored at –80°C
until measurement.
Samples were separated by HPLC using a Kromasil
250mm�4.6mm, 5μm particle size column (Eka Chemicals, AB,
Bohus, Sweden) and its own guard column. Injection volume was
50μl of undiluted extract. HPLC conditions (solvents, gradient, flow
rate, detection) were applied as described in Lazzarino et al.
(Lazzarino et al., 2003). ATP, ADP and ATP standards were
purchased from Sigma. Calibration and calculation of adenylate
concentration in the samples were done using Karat Software 7.0
(Beckman Coulter GmbH, Krefeld, Germany). Energy charge (EC)
was calculated after Atkinson (cf. Ataullakhanov and Vitvitsky,
2002):
EC = (ATP + ADP/2) / (ATP + ADP + AMP) .
The adenylate pool was calculated after Ataullakhanov and Vitvitsky
(Ataullakhanov and Vitvitsky, 2002):
Adenylate pool = ATP + ADP + AMP .
Fig. 1. Transmission electron microscope image of an A. opercularis
adductor muscle with visible mitochondria in the middle-right part of the
picture.
THE JOURNAL OF EXPERIMENTAL BIOLOGY

2495Swimming physiology of aged scallops
Glycogen
Glycogen concentration was determined after Kunst et al. (Kunst
et al., 1984) and Keppler and Decker (Keppler and Decker, 1974).
Muscle tissue (20–50mg) was ground in liquid nitrogen, 1ml ice-
cold Milli-Q water added and the sample sonified on ice at 30%
output control (Branson Sonifier Cell disruptor B15; Danbury, CT,
USA). The homogenate was incubated for 10min at 95°C for protein
denaturation. To hydrolyse glycogen to glucose, 250μl of the
homogenate was mixed with 500μl acetate buffer (0.1mol l–1,
pH4.8) and 20μl amyloglucosidase (Roche, Mannheim, Germany)
and incubated for 2h at 40°C. The rest of the homogenate was kept
on ice for later determination of the free glucose concentration.
After incubation, both samples were centrifuged at 15000g for
10min at 4°C. The supernatant was saved for glucose determination
and measured, using the glucose determination kit (D-glucose UV
test, r-biopharm, Darmstadt, Germany) at 340nm in the photometer.
A standard curve was prepared using the standard solution of the kit.
Glutathione content, intracellular pH, redox potential
The concentrations of glutathione in the oxidised (GSSG) and
reduced (GSH) forms were measured after Fariss and Reed (Fariss
and Reed, 1987), using high performance liquid chromatography
(HPLC). The principle of the measurement is the derivatisation of
the thiols with dinitrofluobenzene (DNFB). GSH oxidation during
extraction is prevented by iodoacetic acid (IAA) binding of GSH.
Tissues were ground in liquid nitrogen and homogenised with
ice-cold perchloric acid (PCA; 10% with 2 mmol l–1
bathophenanthrolinedisulfonic acid; bpDS) at 1/10 (w/v) bubbled
with nitrogen prior use. Following centrifugation at 15000g and
4°C for 5min, 500μl of the supernatant was transferred to a fresh
reaction vial and 10μl of the pH indicator (1mmol l–1 m-Cresol
Purple in H2O with 0.5mol l–1 iodoacetic acid) and 50μl internal
standard (1mmol l–1 gamma-glutamyl-glutamate in 0.3% PCA)
added. Samples were titrated to pH8.5 with 4mol l–1 KOH with
0.3mol l–1 n-morpholinopropanesulfonic acid and incubated for
45min at room temperature to allow iodoacetic acid to bind the
GSH. After 5min centrifugation at 15000g and 4°C, 1% DNFB
(1% 1-fluoro-2.4-dinitrobenzene) in ethanol was added to the
supernatant at a 1:3 ratio and incubated in a dark vial for 24h at
room temperature without shaking. Standards of GSH and GSSG
were prepared in 10% PCA withbpDS and treated as samples.
Prior to injection into the HPLC, thawed samples were again
centrifuged for 1min at 7500g and 4°C, to remove the remaining
PCA and the supernatant filtered through a 0.2μm nylon membrane
filter. Samples were transferred to dark autosampler vials and
injected using an autosampler that was thermostatted to 4°C.
Separation was achieved on a NH2-spherisorb column (240�4mm,
5μm particles) and its own guard column at 39°C using a binary
solvent system of A: 80% methanol–water, and B: 80% solvent A
and 20% acetate stock (272g sodium acetate trihydrate diluted in
122ml water plus 378ml glacial acetic acid). Both solvents were
degassed and filtered (0.45μm pore size) prior to use. Flow rate
was 1.2 ml min–1 at a maximal backpressure of 2500 psi. The
gradient program was: 90% A, 10% B for 12min followed by 30min
of linear gradient elution to 45% A, 55% B and a subsequent 8min
hold. Thereafter the system was returned to the initial conditions
within 5min and re-equilibrated for 15min.
Tissue pH
Tissue pH (pHi) was determined using the homogenate technique
(Pörtner et al., 1990) in a system thermostated at 10°C, the
maintenance temperature of the scallops.
Prior to measurements, the pH electrode (SenTix Mic, WTW,
Weilheim, Germany) was calibrated at the A. opercularis in situ
temperature of 10°C with precise calibration solutions (AppliChem,
Darmstadt, Germany; pH6.865 – A1259; pH7.413 – A1260). The
pH were recorded on a Kipp and Zonen chart recorder.
For tissue measurements, muscle tissue (100–150 mg) was
ground in liquid nitrogen and the powder added to an Eppendorf
tube containing 0.3 ml of medium composed of 160 mmol l–1
potassium fluoride, 2 mmol l–1 nitrilotriacetic acid. The tube was
closed after layering with air-bubble-free medium, and the tissue
homogenised by ultrasound (Branson, sonifier 450, duty cycle
40%, output control 8) at 0°C and centrifuged at 20 000g at 10°C
for 30 s. The pH in the supernatant was determined in the system
thermostatted at 10°C.
Glutathione concentrations and corresponding pHi values of each
sample were used to calculate the tissue redox potential after Schafer
and Büttner (Schafer and Büttner, 2001).
Catalase activity
Catalase activity was determined after Aebi (Aebi, 1984). Frozen
tissue (100–150mg) was ground in liquid nitrogen and homogenised
with a micropistill in 50mmol l–1 phosphate buffer (50mmol l–1
KH2PO4, 50mmol l–1 Na2HPO4, pH7.0) with 0.1% Triton X-100
at 1:5 (w/v). Samples were centrifuged at 13000g for 15min at
4°C. The activity was determined by recording the time of H2O2
decomposition, resulting in a decrease of absorption from 0.45 to
0.4 at 240nm (1 unit).
Lipidperoxidation
Malondialdehyde
Malondialdehyde (MDA) concentrations were measured after
Uchiyama and Mihara (Uchiyama and Mihara, 1978). Muscle tissue
(200mg) was ground in liquid nitrogen and homogenised in a glass
potter with 0.2% phosphoric acid (bubbled with nitrogen) 1:6 (w/v).
The same amount of 2% phosphoric acid was added to achieve a
final concentration of 1.1% phosphoric acid and homogenised again.
0.3ml of the homogenate was transferred to a glass vial and 0.3ml
1% thiobarbituric acid (TBA) solution added (0.5 g TBA in
50mmol l–1 NaOH + 0.5ml 10mmol l–1 BHT + 0.2ml 7% PCA).
As a blank, 0.3ml 3mmol l–1 HCl was added to 0.3ml sample. The
pH of samples and blanks was adjusted to 1.6. Samples were
incubated at 100°C for 60min. After cooling, 1.5ml butanol was
added to each sample and blank and the mixtures vortexed for 40s.
Samples were centrifuged for 5min at 1000g at room temperature.
MDA concentrations were measured in the TBA-containing butanol
phase at 532 and 600nm.
Lipidhydroperoxides
The concentrations of lipidhydroperoxides [LOOH; cumene
hydroperoxides (CHP) equivalents] were measured after Hermes-
Lima et al. (Hermes-Lima et al., 1995), modified for microwell
plates. Muscle tissue (100–200mg) was ground in liquid nitrogen
and homogenised in a glass potter with 100% nitrogen-bubbled
methanol (1:5–1:10w/v) and centrifuged for 5min at 1000g and
4°C. Each microwell contained 75μl FeSO4 (1mmol l–1), 30μl
H2SO4 (0.25 mmol l–1), 30μl xylenol orange (1 mmol l–1) and
160.5μl distilled water. 4.5μl sample was added and incubated for
3h at room temperature and absorbance measured at 580nm (E1).
For blank subtraction, the sample amount was substituted with
distilled water. After determination of E1, 1.5nmol CHP was added
to each sample as an internal standard, and extinction recorded again
after 40min (E2). LOOH concentrations were then calculated as
THE JOURNAL OF EXPERIMENTAL BIOLOGY

2496
CHP equivalents and expressed as mg–1 tissuewetmass, according
to Hermes-Lima et al. (Hermes-Lima et al., 1995).
Statistical analysis
To identify changes of respiration and ROS generation of isolated
mitochondria with size, a linear regression analysis was used
(GraphPad Software). To evaluate the effect of age and groups for
glutathione, adenylates, LOOH and MDA, a full factorial covariance
model (ANCOVA) was used: dependent parameter versus age and
group and age � group. ANOVA and post-hoc tests were applied
to determine significant differences between groups at a significance
level of at least P<0.05 (SAS software JMP 5.0.1a).
For CS and glycogen analysis, a two-tailed unpaired t-test was
used to investigate differences between groups using GraphPad
Software 4.0. If data did not show Gaussian distribution, differences
were tested using the non parametric Mann–Whitney test. Data that
showed different variances were investigated for differences between
groups using the unpaired t-test with Welch’s correction. Outliers
were identified using the ESD method (GraphPad Software). All
data are presented as mean values ± standard errors unless specified
otherwise.
RESULTS
Mitochondrial state 3 and 4 respiration using glutamate as substrate
did not change significantly with size and age in the chosen
size range (51–70mm). A slight decrease could be observed but
was not significant (Fig.2). Mean respiration rates (±s.e.m.) were
10.3 (0.65) nmol O2 mg–1 protein min–1 in state 3 and 4.7
(0.39)nmolO2 mg–1 proteinmin–1 in state 4.
E. E. R. Philipp and others
H2O2 generation rates in state 2 with either succinate (N=5) or
glutamate (N=16) was close to the detection limit with 0.0019
(0.0010)nmolH2O2 mg–1 proteinmin–1 for succinate and 0.0013
(0.0007)nmolH2O2 mg–1 proteinmin–1 for glutamate. After addition
of antimycin and SOD, H2O2 generation increased to values of 0.028
(0.0036)nmolH2O2 mg–1 proteinmin–1 for glutamate respiration and
0.013 (0.003)nmolH2O2 mg–1 proteinmin–1 for succinate – rotenone
respiration.
Mitochondrial volume density was significantly lower in phasic
adductor muscle of bigger compared with smaller individuals
(Table 2), which corresponds perfectly with the lower citrate
synthase activities found in bigger compared with smaller specimens
(Table2).
ADP concentrations and energy charge (EC) were not
significantly different between smaller and bigger individuals but
showed significant differences between the experimental groups
(two-way ANOVA). For further analysis, data of smaller and bigger
individuals were combined for each experimental group (Fig.3).
An increase in ADP and AMP and a decrease in EC were found in
both stress groups (groups 1 and 3) compared with the control (group
0) and recovery groups (group 3 and 4; Fig.3). All parameters
showed either a decrease or an increased during exercise and
returned to control levels after the recovery period (Fig.3). In most
cases, however, the results were not significant because of the high
inter-individual variation in the biochemical response (Fig.3). The
ATP concentration and the overall adenylate pool
did not differ significantly between exercise groups
in smaller or bigger individuals throughout the
swimming experiment, but were overall lower in
bigger (older) than young animals (Fig. 4A,B;
P<0.05).
Glycogen concentrations were significantly
higher in smaller than in bigger individuals of
groups 0 and 4 (Fig.5). Furthermore, glycogen
concentrations decreased significantly in smaller
individuals throughout the swimming experiment
Table2. Mitochondrial volume densities and CS activities of the striated adductor
muscles of the two sizes of scallops
Small/young individuals (N) Big/old individuals (N) P-value*
VV(Mito/Mf) 1.270±0.117 (7) 0.922±0.033 (7) �0.01
CS (i.u. g–1 wet mass) 2.271±0.083 (10) 1.581±0.1082 (10) �0.01
VV(Mito/Mf), mitochondrial volume densities; CS (i.u. g–1 wet mass), citrate synthase activity.
Mean size of scallops: smaller, 49.29±6.02 mm for VV(Mito/Mf) and 51.11±2.2 mm for CS;
bigger, 68.0±1.91 mm for VV(Mito/Mf) and 68.0±1.91 mm for CS.
*Two-sided unpaired t-test.
50 60 70 80
R
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Shell height (mm)
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5
7
9
11
13
15
17
Fig. 2. Respiration of isolated mitochondria of phasic adductor muscle of A.
opercularis in state 3 (black circles) and state 4 (grey circles). Each dot
represents the mean of 1–3 replicate measurements of a single
mitochondrial isolation.
Control 1 min
stress
15 min
recovery
1 min
stress
15 min
recovery
0
0.25
0.50
0.75
ADP AMP
a
a
a
a
Energy charge
0.90
0.95
1.00
a
a a
En
er
gy
c
ha
rg
e
AD
P,
A
M
P
(µm
o
l g
–
1
w
e
t m
a
ss
)
*
*
Fig. 3. Data of energy charge (white squares), ADP (grey squares) and
AMP (black squares) measured in muscle tissue of both the smaller and
bigger individuals, in the different experimental groups (N=12–22). Groups
with the same letter are significantly different from each other (P<0.05). If
more than two groups have the same letter, the group marked with an
asterisk is significantly different to all other groups.
THE JOURNAL OF EXPERIMENTAL BIOLOGY