Metabolic and physiological responses in tissues of the long-lived bivalve Arctica islandica to oxygen deficiency.

ti
b,c,
reme
Received in revised form 9 December 2010
Accepted 14 December 2010
Available online xxxx
al. 2009). Arctica islandica reduces metabolic expenditure, when being
Comparative Biochemistry and Physiology, Part A xxx (2011) xxx–xxx
CBA-09091; No of Pages 7
Contents lists available at ScienceDirect
Comparative Biochemistry
j ourna l homepage: www.eThe long-lived ocean quahog, Arctica islandica, with reported
individual ages ofmore than 350 years (Schöne et al. 2005;Wanamaker
et al. 2008) is among a few species in the Baltic and the North Seawhich
survive frequent and prolonged exposures to hypoxia and anoxia
(Oeschger and Storey, 1993; Diaz and Rosenberg, 1995). The bivalves
colonize pea gravel and muddy bottom sediments and constitute a
major biomass component among the hypoxia tolerant survivor species
in eutrophicated areas (Rosenberg et al. 1992). A low standard
metabolic rate, slow but stable cell turnover rates, and high antioxidant
protection levels that are maintained into old ages up to 200 years
support the extreme longevity of this species (Abele et al. 2008; Strahl
and Abele, 2010).
confronted to environmental hypoxia or anoxia. Thus, caloric heat
production in Baltic Sea specimens fell to 1% of fully aerobic rates after
60 days of experimental anoxia exposure (Oeschger 1990). During
prolonged burrowing and shell closure of N24 h bivalves from the Irish
Sea loweredheart beat to10%of normoxic beat frequency (Taylor1976).
Spontaneous burrowing and shell closure cause hypoxia in mantle
cavity water and are frequently observed in undisturbed A. islandica
maintained in normoxic seawater (Taylor 1976; Abele et al. 2010).
In addition to energy saving behavior, A. islandica is, like other
hypoxia tolerant ectotherms, endowed with a well established
anaerobic energy producing system (Taylor 1976; Livingstone et al.
1983; Oeschger 1990; Oeschger and Storey 1993). Bivalves from theThe capacity of A. islandica to survive oxy
presumably rooted in its energetically econom
⁎ Corresponding author. Alfred-Wegener Institute fo
Building E, Am Handelshafen 12, 27570 Bremerhaven,
1567; fax: +49 471 4831 1149.
E-mail addresses: Julia.Strahl@awi.de (J. Strahl), Dor
1 Tel.: +49 471 4831 1311; fax: +49 471 4831 1149
1095-6433/$ – see front matter © 2011 Elsevier Inc. Al
doi:10.1016/j.cbpa.2010.12.015
Please cite this article as: Strahl, J., et al., Me
deficiency, Comp. Biochem. Physiol. A (201extremely slow growth with low oxygen consumption rates (Begum et1. IntroductionKeywords:
Arctica islandica
Hemocytes
Hypoxia
Anoxia
Metabolic rate depression
Anaerobic metabolism
Glutathioneconditions vs. no oxygen inmantle, gill, adductor muscle and hemocytes of the ocean quahog, specimens from
the German Bight were maintained for 3.5 days under normoxia (21 kPa=controls), hypoxia (2 kPa) or
anoxia (0 kPa). Tissue levels of anaerobic metabolites octopine, lactate and succinate as well as specific
activities of octopine dehydrogenase (ODH) and lactate dehydrogenase (LDH) were unaffected by hypoxic
incubation, suggesting that the metabolism of A. islandica remains fully aerobic down to environmental
oxygen levels of 2 kPa. PO2-dependent respiration rates of isolated gills indicated the onset of metabolic rate
depression (MRD) below 5 kPa in A. islandica, while anaerobiosis was switched on in bivalve tissues only at
anoxia. Tissue-specific levels of glutathione (GSH), a scavenger of reactive oxygen species (ROS), indicate no
anticipatory antioxidant response takes place under experimental hypoxia and anoxia exposure. Highest
specific ODH activity and a mean ODH/LDH ratio of 95 in the adductor muscle contrasted with maximal
specific LDH activity and a mean ODH/LDH ratio of 0.3 in hemocytes. These differences in anaerobic enzyme
activity patterns indicate that LDH and ODH play specific roles in different tissues of A. islandica which are
likely to economize metabolism during anoxia and reoxygenation.
© 2011 Elsevier Inc. All rights reserved.gen limited conditions is
ized lifestyle, combining
Baltic Sea switch
exposure to an
exclusively on gl
(Oeschger 1990)
drogenase (ODH
(LDH) guarantee
a constant suppl
(NADH/NAD+ ra
r Polar and Marine Research,
Germany. Tel.: +49 471 4831
is.Abele@awi.de (D. Abele).
.
l rights reserved.
tabolic and physiological responses in tissues
1), doi:10.1016/j.cbpa.2010.12.015y metabolic and physiological responses to low oxygen
Article history:
Received 21 September 2010In Arctica islandica, a long lifespan is associated with low metabolic activity, and with a pronounced tolerance
to low environmental oxygen. In order to studMetabolic and physiological responses in
islandica to oxygen deficiency
Julia Strahl a,b,1, Ralf Dringen b,c,d, Maike M. Schmidt
a Alfred-Wegner Institute for Polar and Marine Research, Bremerhaven, Germany
b Center for Biomolecular Interactions Bremen, University of Bremen, Bremen, Germany
c Center for Environmental Research and Sustainable Technology, University of Bremen, B
d School of Psychology and Psychiatry, Monash University, Clayton, Australia
e University of Bremen, Bremen, Germany
a b s t r a c ta r t i c l e i n f ossues of the long-lived bivalve Arctica
Silvia Hardenberg e, Doris Abele a,⁎
n, Germany
and Physiology, Part A
l sev ie r.com/ locate /cbpato anaerobiosis during the first 48 h of experimental
oxia and can survive extended periods of time
ycolytic andmitochondrial anaerobic ATP production
. Pyruvate dehydrogenases such as octopine dehy-
), alanopine, strombine and lactate dehydrogenases
the continuous flux of glycolysis and, consequently,
y of ATP by maintaining a low cytosolic redox-status
tio) during anaerobiosis (Gäde and Grieshaber 1986).
of the long-lived bivalve Arctica islandica to oxygen

water depth, using a trawl net. Clams were transported in cooled
containers to the Alfred-Wegener Institute for Polar and Marine
Research in Bremerhaven. Bivalves were held at 10 °C and 33 PSU in
60-l aquaria with recirculating seawater containing 10 cm of gravel
sediment andwere fed once a weekwith amixture of Nannochloropsis
occulato, Phaeodactylum tricornutum and Chlorella sp. (DT's plankton
farm, USA, 3 mL bivalve−1 week−1).
2.2. Materials
Unsterile 96-well plates were purchased from Sarstedt (Karlsruhe,
Germany). Glutathionedisulfide (GSSG) and glutamate phosphate
were obtained from Fluka (Buchs, Switzerland). Octopine, octopine
dehydrogenase, 5-5′-dithio-bis-(2-nitrobenzoic acid), tri-sodium cit-
rate and ethylenediamine tetraacetic acid were purchased from
Sigma (Steinheim, Germany). NADPH, NADH (disodium salt), NAD+,
L-arginine-hydrochloride, sulfosalicylic acid and perchloric acid
(60%) were obtained from Applichem (Darmstadt, Germany). Alpha-
D-glucose was obtained from Serva (Heidelberg, Germany) and
glutamate pyruvate transaminase and GSSG reductase from Roche
(Mannheim, Germany). All other chemicals, in the highest purity
available,werepurchased fromMerck (Darmstadt, Germany) and Fluka.
2.3. Incubation experiments and sample preparation
Arctica islandica were exposed individually in 3-L flasks filled with
natural seawater at constant water temperatures of 10 °C and salinity
of 33 PSU. Three days before the start of the experiment, bivalveswere
Data are presented as mean±SD with n=7-10 per tissue and treatment in (A) and
n=3-7 in (B).
a Significant differences in GSx content of normoxic and hypoxic hemocytes
compared to mantle (Two-way ANOVA Pb0.0001; Bonferroni Pb0.01).
b Significant differences in GSSG (in % of GSx) of normoxic and hypoxic gill compared
to adductor muscle (Two-way ANOVA Pb0.0001; Bonferroni Pb0.05).
c Significant differences in GSSG (in % of GSx) in normoxic hemocytes compared to
adductor muscle (Two-way ANOVA Pb0.0001; Bonferroni Pb0.01).
2 J. Strahl et al. / Comparative Biochemistry and Physiology, Part A xxx (2011) xxx–xxxActivities of all four pyruvate dehydrogenases were detected in the
muscle tissue of A. islandica (Livingstone et al. 1983). LDH activity is
reported to be low inmollusks compared to other invertebrates such as
crustacea or insects,whereas activities of enzymes essential for octopine
and succinate productionarehigh (Livingstoneet al. 1983;Grieshaber et
al. 1994). Maximal lactate concentrations in the hemolymph of five-day
burrowed A. islandica from the Irish Sea amounted to only 5 μg mL−1
(Taylor 1976) compared to 5200 μg mL−1 in crayfish hemolymph after
16 h of anoxic exposure (Grieshaber et al., 1994). No data exist on
octopine or lactate formation in different tissues of the ocean quahog,
but succinate, propionate and acetate accumulate in adductor and foot
muscle of Baltic Sea A. islandica after two days of experimental anoxia
(Oeschger 1990).
A yet unresolved question is whether there is a difference between
environmental hypoxia and anoxia for the metabolic and physiological
response ofA. islandica.Mostly anoxia is simply regarded as an “extreme
case of environmental hypoxia” but, indeed, this may not be so for
extremely hypoxia tolerant and yet basically aerobic species such as A.
islandica. So, is there a low tissue PO2 threshold in hypoxia tolerantmud
clams down to which aerobic energymetabolism can be maintained by
economizing energy expenditures, and below which the animals need
to switch to anaerobiosis? Taylor (1976) found that the mantle cavity
water of five-day burrowed A. islandica still contained low quantities of
oxygen and had a PO2 of 1.3 kPa. As the accumulation of anaerobic
metabolites was not determined in his study, it is not clear whether the
mitochondria were working at very slow aerobic rates or had already
started anaerobic energy production.
Maintaining themitochondria in a slowperformingandaerobic state
and, thereby, avoiding the complete reduction of electron transport
chain intermediates, could support longevity in A. islandica, as these
reduced mitochondrial complexes are held responsible for the en-
hanced formation of ROS during reoxygenation. Cellular ROS aremainly
generated during mitochondrial respiratory activity (Turrens 2003;
Balaban et al. 2005) and the conversion of oxygen to H2O2 in
invertebrate mitochondria is usually less than 1% of the oxygen
consumed under phosphorylating state III conditions (Abele et al.
2007). The absolute amount of cellular ROS production in marine
invertebrates depends on tissue oxygenation (Buchner et al. 2001) as
well as on the number and the site specific redox potential of
mitochondrial chain units (Balaban et al. 2005). Thus, our a priori
assumption is thatROSproduction shouldbe low in tissuesofA. islandica
under normoxia (see also Buttemer et al. 2010, Table 1) and even lower
in ametabolically down-regulated state, inwhichelectron transport and
oxidative phosphorylation are slow but functioning. Nevertheless, the
antioxidant defence system, including the synthesis of ROS-scavenging
glutathione (GSH), may be up-regulated during MRD in A. islandica to
preventoxidative stressduring reoxygenation. This has beendetected in
several hypoxia- and anoxia-tolerant invertebrates undergoing estiva-
tion and freezing (Hermes-Lima and Zenteno-Savín, 2002).
The aim of the present study is the investigation of tissue-specific
responses of A. islandica to severe hypoxia and anoxia. In order to
compare the activity of LDH and ODH in tissues and hemocytes, to
measure the contents of anaerobic metabolites and of GSH as well as
to determine the cellular thiol redox state, bivalves from the German
Bight were incubated for 3.5 days under normoxic, hypoxic or anoxic
conditions. Oxygen consumption was measured in isolated gills of A.
islandica pre-incubated at normoxic or hypoxic PO2, in order to
confirm the effect of diminishing oxygen availability onmetabolic rate
in the organ most directly exposed to environmental oxygenation.
2. Materials and methods
2.1. Bivalve collection and maintenance
Arctica islandica were collected in May 2008 at Helgoland “Tiefe
Rinne” in the German Bight (54°09.05′N, 07°52.06′E) at 40−45 m
Please cite this article as: Strahl, J., et al., Metabolic and physiological res
deficiency, Comp. Biochem. Physiol. A (2011), doi:10.1016/j.cbpa.2010.Table 1
(A) Contents of GSx and GSSG in freshly dissected tissues and hemocytes of Arctica
islandica that had been incubated under normoxic or hypoxic conditions and (B) contents
of GSx in tissues of bivalves that had been incubated under normoxic and anoxic
conditions. The latter were stored at -80 °C before measurement.
A
GSx
(nmol mg-1 protein)
GSSG
(nmol mg-1 protein)
GSSG
(% of GSx)
Normoxic (21 kPa)
Mantle 1.90±0.90 0.41±0.22 20±9
Gill 5.51±6.15 1.69±2.15 26±12b
Adductor muscle 4.82±4.85 0.56±0.82 10±6
Hemocytes 7.94±5.87a 1.64±1.06 28±16c
Hypoxic (2 kPa)
Mantle 1.29±0.49 0.12±0.08 9±4
Gill 3.20±0.36 0.77±0.19 24±5b
Adductor muscle 2.68±1.77 0.14±0.05 7±5
Hemocytes 6.09±3.35a 0.78±0.64 13±8
B
GSx
(nmol mg-1 protein)
Normoxic (21 kPa)
Mantle 3.05±0.05
Gill 9.00±0.18
Adductor muscle 3.43±0.34
Anoxic (0 kPa)
Mantle 4.09±1.77
Gill 10.30±3.92
Adductor muscle 5.24±3.87not fed any more to avoid eutrophication through faeces and
ponses in tissues of the long-lived bivalve Arctica islandica to oxygen
12.015

3J. Strahl et al. / Comparative Biochemistry and Physiology, Part A xxx (2011) xxx–xxxmicrobial contamination of the experimental water. Arctica islandica
were incubated under normoxic conditions (21 kPa) or under hypoxic
conditions (2 kPa) by using a gas-mixture of nitrogen and oxygen (Air
liquid, Germany) or under complete anoxia (0 kPa). Siphon-status of
each individual was visually checked and recorded 3 times per day
during incubations. After 3.5 days of incubation, shell closure of each
bivalve was prevented by inserting a metal bar, 3 mm thick and 3 cm
long. Then hemolymph was taken from the adductor muscle of each
individual with a sterile needle and a 10 mL-syringe to determine
enzyme activities and concentrations of anaerobic metabolites.
Subsequently bivalves were dissected. One fresh sample-set of
mantle, gill, adductor muscle and hemocytes was used to determine
enzyme activities and the contents of total glutathione (GSx=GSH+
2× GSSG) and of GSSG. A second sample-set of each tissue type and of
hemolymphwithout hemocytes was snap frozen in liquid nitrogen for
laboratory analysis of anaerobic metabolites. The third group of
bivalves was incubated under complete anoxia in order to confirm
that our hypoxic incubation period covered the timewindow inwhich
anaerobicmetabolism can be induced in German Bight A. islandica and
to test for octopine, lactate and succinate accumulation. Samples of
under normoxia- and anoxia-incubated bivalves stored at −80 °C
were further analyzed for the specific GSx content.
2.4. Determination of LDH and ODH
Freshly dissected mantle, gill and adductor muscle samples of
bivalves incubated under normoxia or hypoxia were homogenized on
ice with an Ultra-Turrax (IKA-Werke, Germany) after adding a 6-fold
volume (w/v) of homogenizing-buffer (20 mM Tris–HCL, 1 mM EDTA,
1 mM DTT, pH 7.5). The homogenate was centrifuged for 15 min at
12,000 g and 4 °C. Fresh hemolymphwas transferred into a 10 mL-tube
and centrifuged for 10 min at 500 g and 4 °C. The pellet containing the
hemocytes was dissolved in 20 mM KPi buffer and incubated on ice for
10 min. The sample was centrifuged for 1 min at 12,000 g and 4 °C and
lactate dehydrogenase (LDH; EC 1.1.1.27) and octopine dehydrogenase
(ODH; EC1.5.1.11) activitieswere determined in supernatants of tissues
and hemocytes according to Livingstone et al. (1990). The absorbance
decrease of NADH at 340 nmwas recorded over 10 min in 15 s intervals
in amicroplate reader (Sunrise, Tecan, Germany). Reactionswere run in
triplicate in the absence and presence of L-arginine to calculate the ODH
activity by subtracting the LDH activity from the total enzyme activity.
2.5. Determination of octopine, lactate and succinate
Frozen mantle, gill and adductor muscle samples of bivalves
incubated under normoxia, hypoxia or anoxia were homogenized on
ice with an Ultra-Turrax and ultrasound (Bandelin, Germany) after
adding a 6-fold volume (w/v) of 0.5 M perchloric acid. The
homogenate was centrifuged for 15 min at 12,000 g and 4 °C and
the supernatant of each sample was neutralized with 2 M KOH and
centrifuged for 5 min at 12,000 g and 4 °C. Octopine and lactate
contents of all tissue samples and of defrosted hemolymph, mantle
cavity water and incubation water samples were determined after
Luisi et al. (1975) and Schmidt and Dringen (2009), respectively. The
reaction mixture for octopine quantification contained 0.25 U of ODH
per well (180 μL), that for lactate quantification 7.15 U LDH per well.
After incubation for 90 min at room temperature (RT) the absorbance
of NADH was measured at 340 nm in the microplate reader. In the
adductor muscle samples additionally the succinate content was
determined after Michal et al. (1976) using a succinic acid assay kit
(Cat. No. 10 176 281 035, Boehringer Mannheim/R-Biopharm,
Germany). The absorbance of NADH was recorded in UV-DU 800
spectrophotometer (Beckmann, Germany) at 340 nm and 37 °C. The
incubation time was prolonged to 30 min to allow complete reaction
of all the succinate in the sample. Contents of metabolites are shown
per mg wet mass (WM).
Please cite this article as: Strahl, J., et al., Metabolic and physiological res
deficiency, Comp. Biochem. Physiol. A (2011), doi:10.1016/j.cbpa.2010.2.6. Determination of glutathione
Freshly dissected mantle, gill and adductor muscle samples of
specimens incubated under normoxia and hypoxia, as well as frozen
samples of bivalves incubated under anoxia, were homogenized on ice
using an Ultra-Turrax and ultrasound in a 6-fold volume (w/v) of 1%
sulfosalicylic acid (SSA). Samples were centrifuged for 1 min at
12,000 g and 4 °C. One milliliter of fresh hemolymph of each of the
individuals incubated under normoxia or hypoxia was centrifuged for
10 min at 500 g and 4 °C, respectively. The pellet containing the
hemocytes was diluted in 100 μL of 1% SSA, incubated on ice for
10 min and centrifuged for 1 min at 12,000 g and 4 °C. The contents of
GSx and GSSG in the supernatants of tissues and hemocyte lysates
were determined as described previously (Dringen and Hamprecht,
1996) in microtiter plates according to the colorimetric method
originally described by Tietze (1969). The detection limit of this assay
is about 0.2 nmol GSx per 500 μL lysate. Specific GSx and GSSG
contents were obtained by normalizing the GSx and GSSG contents of
the tissue supernatants to the protein content of the respective acid
precipitated pellets of tissues and hemocyte samples.
2.7. Determination of protein content
The pellets of tissues and hemocyte samples used for the
determination of GSx were air-dried for 30 min, homogenized with
ultrasound in 0.5 M NaOH and incubated for 2 h at RT. Protein
contents of these homogenates as well as of the homogenates used for
ODH and LDH enzyme activity assays were determined according to
the method of Lowry et al. (1951) using bovine serum albumin as a
standard.
2.8. Measurement of gill respiration rates at three different PO2
Bivalves were kept without food for three days before the
experiment to eliminate the impact of specific dynamic action on gill
respiration (Bayneet al. 1976). Gills ofA. islandicawere freshly dissected
and transferred in cooled respiration-buffer (450 mMNaCl, 10 mMKCl,
20 mM MgCl2, 10 mM HEPES, 1 mM EGTA, 0.5 mM DTT, 0.055 mM
glucose, pH 7.4). Gill filaments were cut into three pieces of 15–25 mg
WM and incubated for 2 h in respiration-buffer at 8 °C and with PO2
adjusted to 2 kPa, 5 kPa or 21 kPa, respectively. Low PO2 of 2 kPa and
5 kPa were adjusted with gas-mixtures of nitrogen and oxygen.
Following incubation, each piece of gill was transferred into a
refrigerated respiration chamber filled with 1.1 mL respiration-buffer
at 8 °C and adequate PO2. Each chamber was equipped with a net fixed
on a plastic ring to protect the gill from themagnet stirrer at the bottom
of the chamber and the chambers were closed hermetically with a
stopper. The oxygen consumption rates of gill piecesweremeasured for
30 min at 2 kPa, 5 kPa or 21 kPa using single channel Microx TX-3
oxygen meters equipped with oxygen needle-optodes (PSt1-L5-TF,
Precision Sensing GmbH, Germany) which were introduced into the
chamber through a borehole in the stopper. Prior to measurements,
optodes were calibrated to 100% air saturation with aerated seawater
and to 0% using a saturated ascorbate solution. After respiration
measurements the WM of each gill piece was determined and
respiration rates were calculated as nmol O2 min−1 mL−1 mg−1WM.
2.9. Statistical analyses
Statistical analyses were performed with GraphPad Prism 5
Software (La Jolla, CA, USA). All data sets were tested for normality
(Kolmogorov–Smirnov test) and homogeneity of variances (Bartlett's
test) before testing for tissue and PO2 specific differences in enzyme
activities, accumulation of anaerobic metabolites, GSx content or gill
respiration.
ponses in tissues of the long-lived bivalve Arctica islandica to oxygen
12.015

3. Results
3.1. Siphon behavior, enzyme activities and anaerobic metabolites in
tissues and hemocytes or hemolymph of A. islandica
All animals survived 3.5 days of experimental exposure to hypoxia
and anoxia. We observed their siphons to be permanently open
during hypoxic and anoxic incubation, whereas bivalves exposed to
fully normoxic oxygenation alternately opened and closed siphons.
Neither LDH nor ODH activities in mantle, gill, adductor muscle
and hemocyte cells differed significantly under hypoxic compared to
normoxic incubation conditions (Fig. 1A, B). However, tissue-specific
differences in the ratio of both enzymes were observed. The adductor
muscle had significantly higher ODH activity than all other tissues
(Fig. 1B) and the highest ODH/LDH ratio (normox/hypox=95.1±
145.4), whereas enzyme ratios in mantle (normox/hypox=1.1±
0.9), gill (normox/hypox=0.3±0.3) and hemocytes (normox/
hypox=0.3±0.3) were significantly lower under normoxic and
hypoxic conditions (Kruskal–Wallis Pb0.0001; Dunns Pb0.05).
Hemocytes had significantly higher LDH activity than the other
tissues (Fig. 1A), and both, LDH and ODH activities were lowest in the
mantle (Fig. 1A, B).
Among the tissues investigated, octopine was only detectable in
the adductor muscle and amounted to 10.5±5.1 pmol mg−1 WM
under normoxic conditions (n=10), 9.4±5.3 pmol mg−1 WM under
hypoxic conditions (n=7) and 9.1±6.5 pmol mg−1 WM under
anoxic conditions (n=10), indicating that 3.5 days of hypoxic or
anoxic exposure do not accelerate octopine formation in A. islandica.
Succinate contents remained unaltered in adductor muscle between
clams exposed to normoxia or hypoxia, but were significantly
found for adductor muscle under hypoxic and normoxic conditions,
and in normoxic hemocytes compared to that of the adductor muscle
(Table 1A).
3.3. Respiration rates of isolated gills
Gill respiration rates of A. islandica decreased significantly with
declining oxygen concentrations in the incubation medium between
21 kPa and 2 kPa (Fig. 3). At 5 kPa gill respiration was 33% lower and
at 2 kPa 60% lower compared to oxygen consumption rates under
normoxia (Fig. 3).
4. Discussion
4.1. Metabolic response to 3.5 days of hypoxia in A. islandica
Experimental hypoxia elicited behavioral and physiological
responses in German Bight A. islandica. Siphons were permanently
4 J. Strahl et al. / Comparative Biochemistry and Physiology, Part A xxx (2011) xxx–xxxFig. 1. Activities of lactate dehydrogenase (LDH) (A) and octopine dehydrogenase
(ODH) (B) in mantle (MA), gill (G), adductor muscle (AM) and hemocytes (HC) of
Arctica islandica incubated under normoxic conditions with a PO2 of 21 kPa (open bars)
or under hypoxic conditions with 2 kPa (filled bars), mean±SD, n=7–10 per tissue
type and treatment. ** LDH activity in hemocytes differs significantly from that of
all tissues under normoxic and hypoxic conditions (two-way ANOVA Pb0.0001;
Bonferroni Pb0.01). *** ODH activity in adductor muscle differs significantly from
that of hemocytes and of all other tissues under normoxic and hypoxic conditions
(two-way ANOVA Pb0.0001; Bonferroni Pb0.001).
Please cite this article as: Strahl, J., et al., Metabolic and physiological res
deficiency, Comp. Biochem. Physiol. A (2011), doi:10.1016/j.cbpa.2010.elevated after the same period of anoxia (Fig. 2). The lactate content
remained below the detection limit in all tissues including hemo-
lymph and in the incubation water under normoxic, hypoxic and
anoxic conditions.
3.2. Glutathione content in tissues and hemocytes of A. islandica
The specific GSx content of mantle, gill, adductor muscle tissue and
hemocytes of A. islandica was almost identical between normoxic and
hypoxic conditions but showed tissue-specific differences that ranged
between 1.3 and 8 nmol mg−1protein (1A). Sample storage at−80 °C
for several months had no effect on GSx content in tissues of normoxic
animals, however GSH was more oxidized after storage in the freezer,
so that the GSSG content cannot be compared between freshly
dissected and stored samples (data not shown). Similar specific GSx
contents than in freshly dissected tissues were found in frozenmantle,
gill and adductor muscle samples stored at−80 °C without significant
differences between bivalves incubated for 3.5 days under normoxia
or anoxia (Table 1B). GSSG amounted to between 7 and 28% of the GSx
determined under normoxic and hypoxic conditions (Table 1A). In gill
and adductor muscle, GSSG content did not differ under both
conditions, while hypoxic exposure reduced the percentage of GSSG
in mantle and hemocytes by about 50% compared to normoxic
exposure (Table 1A), although this difference did not reach the level
of significance. Hemocytes had higher GSx contents than the other
tissues under normoxia and hypoxia, followed by GSx content in the
gills, but the observed differences were statistically significant only
compared to the GSx contents of the mantle. The percentage of
GSSG in GSx was significantly higher in gills compared to the values
Fig. 2. Succinate content in the adductor muscle of Arctica islandica incubated under
normoxic conditions with a PO2 of 21 kPa, hypoxic conditions with 2 kPa and anoxic
conditions with 0 kPa, mean±SD, n=7–10 per treatment. ** Significant differences
between anoxic compared to normoxic and hypoxic conditions (one-way ANOVA
Pb0.0001; Bonferroni Pb0.01).open at 2 kPa and respiration rates in gills decreased more rapidly
ponses in tissues of the long-lived bivalve Arctica islandica to oxygen
12.015

5J. Strahl et al. / Comparative Biochemistry and Physiology, Part A xxx (2011) xxx–xxxbelow than above 5 kPa of environmental oxygen. Oxyconform
respiration rates (=reduced oxygen consumption with decreasing
PO2) and a similar critical PO2 of 4.9 kPa were already determined in
respiration experiments with isolated mantle tissue of Baltic Sea A.
islandica (Tschischka et al. 2000). This indicates that MRD sets in
below 5 kPa in the ocean quahog to idle on a reduced but oxygen-
based metabolic level down to extremely low environmental PO2.
Consequently, 3.5 days of hypoxic incubation did not affect the tissue
levels of the anaerobic metabolites, and the specific activities of LDH
or ODH were not up-regulated during hypoxic exposure. In contrast,
succinate accumulated in the adductormuscle of clams exposed to the
same period of anoxia. Succinate is the first intermediate which
signals the onset of anaerobicmetabolism in A. islandicamuscle tissue,
and our results are in line with those of Oeschger (1990), who found
around 55 nmol succinate mg−1 WM in the adductor muscle of Baltic
Sea A. islandica after 3.5 days of anoxic incubation.
The reduction of metabolic oxygen demand below 5 kPa enables
the ocean quahog to maintain tissues and mitochondria aerobic down
to a PO2 of at least 2 kPa. A similar response has been recorded in
starved Abra tenuis, where direct calorimetry and respiration mea-
surements with whole animals documented a fully aerobic metabo-
lism under hypoxic conditions N2.3 kPa of oxygen and the onset of
anaerobic metabolism below 2.3 kPa (Wang and Widdows, 1993).
Permanently open siphons of A. islandica observed in our 3.5-days
hypoxic incubation, suggest that the animals increase ventilation to
remain aerobic during severe oxygen limitation. Similarly, Brand and
Taylor (1974) found A. islandica from the Irish Sea to increase the
proportion of time spent on active pumping of water to 95% at 5 kPa
PO2, whereas at normoxic conditions each pumping period was
Fig. 3. Respiration rate of isolated Arctica islandica gill tissue incubated at normoxic PO2
of 21 kPa, 5 kPa and hypoxic PO2 of 2 kPa, mean±SD, n=9–16 per treatment.
* Significant difference between gill respiration at 5 kPa and 21 kPa (Kruskal–Wallis
Pb0.0001, Dunn's Pb0.05). ** Significant difference between gill respiration at 2 kPa
and 21 kPa (Kruskal–Wallis Pb0.0001, Dunn's Pb0.001).followed by a ventilatory pause lasting for several minutes. In keeping
with the better exploitation of low environmental PO2, mitochondrial
phosphorylation efficiency of White Sea A. islandica was found to be
significantly higher at low experimental oxygen conditions of 7.3 kPa
than at normoxic PO2 (Tschischka et al. 2000). These physiological
adaptations suggest that the critical PO2 (Pcrit) in hypoxia tolerant
ocean quahog marks the onset of MRD and energy saving, rather than
the compensation of oxygen deficiency by anaerobiosis, as proposed
for other species (Grieshaber et al. 1994; Boutilier and St-Pierre, 2000;
Boutilier 2001). We assume that shifting the onset of anaerobiosis to
tissue PO2 values far below Pcrit forms part of the hypoxia-tolerance-
strategy of A. islandica to survive prolonged periods of environmental
oxygen deficiency.
4.2. Tissue-specific antioxidant protection at normoxic and hypoxic
conditions
Our next question addressed the cellular antioxidant system and
possible ROS-formation in tissues and hemocyte cells of A. islandica
Please cite this article as: Strahl, J., et al., Metabolic and physiological res
deficiency, Comp. Biochem. Physiol. A (2011), doi:10.1016/j.cbpa.2010.under hypoxic conditions. Metabolic depression in A. islandica does
not only play an important role in energy reduction, it also represents
a protective mechanism to slow mitochondrial ROS production and
thus may be a life-prolonging strategy in the ocean quahog (Abele
et al. 2010). The ratio of GSSG to GSx was lower at 2 kPa compared to
21 kPa in mantle and hemocytes, which indicates that less ROS are
produced at hypoxic conditions. This is in keeping with Boyd and
Burnett (1999), who found that ROS production rates of oyster
hemocytes under hypoxia (PO2=1.47 kPa) amount to only 33% of the
normoxic rates (PO2=5.2 kPa).
The total GSx content in tissues of the ocean quahog indicates
constant GSH redox cycling and/or synthesis of GSH during normoxia,
hypoxia and anoxia. Thus no anticipatory antioxidant response to a
possible reoxygenation event after environmental oxygen shortage
took place. In line with this, antioxidant enzyme activities
remained constant in the mantle and gill of metabolically depressed
A. islandica after 24 h of 0 kPa in the mantle cavity water and after
3.5 days of burrowing (Strahl et al., submitted for publication).
Higher levels of free radical damage during awakening and tissue
reoxygenation (Ramos-Vasconcelos and Hermes-Lima 2003, Hermes-
Lima and Storey 1995), as well as an up-regulation of antioxidant
capacities are reported for estivating and hibernating invertebrates
(Hermes-Lima and Zenteno-Savín, 2002, Bickler and Buck, 2007).
In contrast a ROS-burst was absent in isolated gill tissue of Iceland
A. islandica (Strahl et al., submitted for publication) and of German
Bight A. islandica (S. Hardenberg, unpublished data) after hypoxia-
reoxygenation. This is possibly due to an alternative oxidase in
A. islandica mitochondria which has a higher P50 than cytochrome-c-
oxidase and provides a phosphorylation-independent bypass for
electrons when cytochrome-c-oxidase capacities are limiting. In
lowering tissue PO2 and minimizing reduction of respiratory chain
intermediates, the alternative oxidase reduces the risk of ROS
formation on reoxygenation in hypoxia tolerant species (Tschischka
et al., 2000). However, so far no molecular confirmation for the
existence of an alternative oxidase in A. islandica exists. The trend of
somewhat higher GSx levels in tissues of anoxia-incubated bivalves,
especially in the gills, is not statistically significant and may reflect
inter-individual variations in GSx.
Bivalves extract oxygen from the seawater mainly via the gill
surface (Bayne et al. 1976), suggesting the possibility of higher ROS
generation in the gills compared to other tissues. Further tissue
respiration rates and phosphorylation efficiency are higher in gills
(Strahl et al., submitted for publication) compared to mantle of
A. islandica (Tschischka et al. 2000), which indicate higher tissues-
specific metabolic activity and explain the need for higher GSx levels
and the occurrence of high GSSG amounts in the gills of the ocean
quahog. Comparably high GSx contents ranging from about 0.6 to
20 nmol mg−1 proteinwere found in gill tissue ofMytilus edulis (Ribera
et al. 1991), Mytilus galloprovincialis (Regoli and Principato, 1995; Lima
et al. 2007) and Perna viridis (Cheung et al. 2001).
In A. islandica equally high contents to gill-GSx and -GSSG were
found in the hemocytes, the bivalves' immune cells. These cells are
capable of performing an oxidative burst to kill and phagocytose
invading pathogens (Boyd and Burnett, 1999; Pruzzo et al. 2005). In
order to minimize potential oxidative damage to adjacent tissues and
cells, antioxidant protection should be high in hemocyte cells. This
hypothesis is in line with results reported by Pipe et al. (1993), who
detected high activities of the antioxidant enzymes superoxide
dismutase and catalase in granular hemocytes of M. edulis. Mean
glutathione levels in hemocyte lysates of the scallop Pecten maximus
were about 13.9 nmol mg-1protein (Hannam et al. 2010) and, thus,
comparable to the levels in A. islandica.
The mantle seems to be metabolically less active than other
investigated tissues of the ocean quahog, because here anaerobic
enzyme activities and GSx content were lowest. Significantly lower
antioxidant enzyme activities in mantle than in foot muscle were also
ponses in tissues of the long-lived bivalve Arctica islandica to oxygen
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