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Differential responses of juvenile and adult South African abalone (Haliotis midae

Linnaeus) to low and high oxygen levels


Andre Vosloo , Anl Laas, Dalene Vosloo
School of Life Sciences, University of KwaZulu-Natal, Private Bag X54001, Durban, 4000, South Africa
a b s t r a c t a r t i c l e i n f o
Article history:
Received 16 May 2012
Received in revised form 4 September 2012
Accepted 4 September 2012
Available online 10 September 2012
Keywords:
Haliotis midae
Abalone
Oxidative stress
Physiology
Comet assay
Hsp 70
Catalase
Marine invertebrates have evolved multiple responses to naturally variable environmental oxygen, all aimed
at either maintaining cellular oxygen homeostasis or limiting cellular damage during or after hypoxic or
hyperoxic events. We assessed organismal (rates of oxygen consumption and ammonia excretion) and cellu-
lar (heat shock protein expression, anti-oxidant enzymes) responses of juvenile and adult abalone exposed to
low (~83% of saturation), intermediate (~95% of saturation) and high (~115% of saturation) oxygen levels for
one month. Using the Comet assay, we measured DNA damage to determine whether the observed trends in
the protective responses were sufcient to prevent oxidative damage to cells. Juveniles were unaffected by
moderately hypoxic and hyperoxic conditions. Elevated basal rates of superoxide dismutase, glutathione per-
oxidase and catalase were sufcient to prevent DNA fragmentation and protein damage. Adults, with their
lower basal rate of anti-oxidant enzymes, had increased DNA damage under hypoxic and hyperoxic condi-
tions, indicating that the antioxidant enzymes were unable to prevent oxidative damage under hypoxic
and hyperoxic conditions. The apparent insensitivity of juvenile abalone to decreased and increased oxygen
might be related to their life history and development in algal and diatom biolms where they are exposed to
extreme diurnal uctuations in dissolved oxygen levels.
2012 Elsevier Inc. All rights reserved.
1. Introduction
Temperature and oxygen are the most important environmental
factors that govern biological processes in nature. Temperature, for
instance, may directly or indirectly drive biogeographical patterns
(Prtner and Knust, 2007). Fitness of species is diminished at temper-
ature and oxygen extremes, which may lead to (a) decreased popula-
tion numbers due to inefcient production (growth and recruitment)
at the edges of the distribution range and (b) eventual exclusion of
species outside specic temperature and oxygen ranges. Just as natu-
ral temperature and oxygen ranges inuence production on the bio-
geographical scale, temperature and oxygen proles of intensive
aquaculture systems determine production and, in this sense, nan-
cial viability of operations.
Oxygen is naturally variable in near-shore marine systems, for in-
stance in Mobile Bay (Gulf of Mexico) (Johnson et al., 2009). Along its
natural Western and Southern distributions, Haliotis midae can be ex-
posed to temporary bouts of anoxia or hypoxia resulting from the
decay of algal blooms. In recent history, rock lobster (Jasus lalandii)
strandings in the south Western Cape Province were driven by oxy-
gen depletion subsequent to a large Ceratium furca bloom and decay
(Pitcher and Calder, 2000). Unlike lobsters, the less mobile H. midae
would be unable to escape these conditions and consequently have
to initiate adaptive responses to the hypoxic conditions.
In the diatom biolm where post-larval abalone settle and devel-
op for roughly three months, oxygen may vary even more drastically.
In the diffusive boundary layer, oxygen may reach 440% of air satura-
tion due to photosynthesis, but may be depleted during dark phase
photosynthesis (Daume, 2006; Roberts et al., 2007). Due to their
small size and limited mobility, larval and juvenile abalone cannot es-
cape these extreme diurnal oxygen uctuations. After growing out of
the diatom biolm, juvenile abalone in the intertidal zone would nat-
urally be exposed to air on a regular basis.
In aquaculture systems, dissolved oxygen in the water is a result of
oxygen utilization (e.g. biomass, stocking density) and oxygen supply
(e.g. water ow rate, aeration efciency, temperature-dependent ox-
ygen solubility). Dissolved oxygen concentrations may uctuate be-
tween 8.2 and 7.2 mg O
2
L
1
in abalone aquaculture systems in
South Africa, and depend on the distance from the tank inlet and
time of day (Yearsley, 2007). Water ow may also decrease or cease
during periods of power outages, leading to temporary decreases in
dissolved oxygen. Farm management interventions e.g. tank/raceway
cleaning and routine weighing and redistribution of biomass, result in
animals being removed from water, spending variable amounts of
time in air, with resultant oxygen supply limitation (Vosloo et al.,
2008). This periodic air exposure may limit the oxygen extraction ef-
ciency of the abalone respiratory system, as adult H. midae occurs
naturally in crevices and on shallow reefs, but reach maximum
Comparative Biochemistry and Physiology, Part A 164 (2013) 192199
Corresponding author. Tel.: +27 312601337.
E-mail address: vosloo@ukzn.ac.za (A. Vosloo).
1095-6433/$ see front matter 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.cbpa.2012.09.002
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Comparative Biochemistry and Physiology, Part A
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densities permanently inundated in kelp (Ecklonia maxima) forests
(Branch et al., 2008).
Although reactive oxygen species (ROS) are formed during normal
mitochondrial processes, ROS levels may increase if there is a mismatch
between oxygen supply and demand. The accurate matching of cellular
oxygen supply and demand can be achieved by attenuating either or-
ganismal energy supply or energy demand, which would limit ROS for-
mation. Energy demand (and hence oxygen consumption) can be
adjusted by decreased reliance on a hierarchy of high-demand cellular
processes like RNA and protein synthesis, ion transport, gluconeogene-
sis and proton leak (Bishop et al., 2002; Staples and Buck, 2009). In
mammals this attenuation is highly ROSdependent (Brookes et al.,
2004).
Biological systems have complex mechanisms to prevent ROS-
induced damage (Lurman et al., 2007a). It has been suggested that
this is a hold-over of the oxygen pollution of the late Cambrian,
when ambient oxygen levels increased (Melzner et al., 2007c). Pro-
gressive lines of defence are (a) the presence of ROS scavenging agents
(e.g. ascorbate, -tocopherol) and inducible proteins such as transferrin
and metallothionein, (b) anti-oxidant enzymes superoxide dismutase
(SOD), glutathione peroxidase (GPx) and catalase (CAT) that enzymati-
cally detoxify ROS to prevent their interference with cellular components
and(c) the cell's natural repair systems canbe inducedto repair damaged
proteins or DNA (Manduzio et al., 2005; Lurman et al., 2007a). Should
ROS levels exceed the rate of scavenging, detoxication and repair, oxida-
tive stress as measured by ROS induced damage to proteins, lipids and
DNA will manifest.
From the above discussions it is clear that the environmental con-
ditions for post-larval, juvenile and adult abalone differ markedly in
terms of oxygen content, leading to the hypothesis that their re-
sponses to increased or decreased ambient oxygen would differ. The
aim of the study reported here was to assess the extent to which ab-
alone, with their relatively primitive respiratory system, are able to
deal with moderately increased or decreased oxygen saturation. Re-
sponses included organismal level responses (oxygen consumption,
ammonia excretion), substrate utilization (O:N ratio, haemolymph
glucose and tissue protein levels), indicators of gas exchange (D-
lactate, haemocyanin concentration), antioxidant protection (SOD,
GPx and CAT activity), cellular repair processes (hsp70 proteins) and
ROS-induced damage (DNA damage as assessed by the Comet assay).
2. Materials and methods
2.1. Pre-experimental acclimation
Juvenile abalone (Haliotis midae; N=450; 12.20.27 g; 41.4
0.27 mm shell length) and adult abalone (N=382; 53.60.24 g;
65.320.15 mm shell length) were moved from the grow-out plat-
forms to an on-farm laboratory two weeks prior to the onset of the ex-
posures (Irvin & Johnson Abalone Division, Danger Point, South Africa).
Filtered, UV treated sea water was circulated through the tanks with a
water ow rate of approximately 20 L h
1
ensuring one water ex-
change in the containers every hour. Water temperature was regulated
at 16 C by a central heater/cooler and oxygen concentration was regu-
lated at 7.570.002 mg L
1
O
2
(means.e.m.) by aeration. The hold-
ing tanks were cleaned twice a week and animals were provided with
0.015 g articial abalone feed (Abfeed S34, Marifeed (Pty) Ltd) per
gram wet body mass.
2.2. Experimental exposure
Both large and small animals were exposed to low (about 80% satu-
ration), intermediate and high (about 120% saturation) oxygen levels
for one month (see Table 1 for details). In the interest of consistency
and ease of reading, these treatments will be referred to as hypoxia,
normoxia and hyperoxia in the rest of the paper. The intermediate/
normoxic oxygen levels were used as a control and atmospheric air
was bubbled through the water. Higher and lower than saturation oxy-
gen levels were maintained by bubbling atmospheric air supplemented
with pure oxygen and nitrogen respectively through the water. Water
temperature was regulated at 16 C. Water temperature and oxygen
levels were measured in the morning and afternoon each day (YSI556
Multiparameter System) and adjustments to the oxygen or nitrogen
ow were made when necessary (Table 1). Previous studies on green
lip abalone (Haliotis laevigata) used similar oxygen concentrations
(Harris et al., 1999).
2.3. Sampling
After one month of exposure, haemolymph was sampled from the
pedal sinus with a one millilitre insulin syringe with a 27 gauge nee-
dle. Gill and muscle tissues were sampled after shucking the animals.
Haemolymph and tissue samples were then snap-frozen in liquid ni-
trogen and transported in dry ice to the University of KwaZulu-Natal
for analysis. Haemolymph samples for the Comet assay were kept on
ice until analysis.
2.4. Physiological parameters
Mass specic oxygen consumption rate of individual animals was
measured in closed systems using sealed respirometers. Respirometer
volumes were adjusted between 200 and 660 mL, and sealed times
between 10 and 110 min, depending on the size of the abalone. This
approach prevented excessive oxygen content decrease during mea-
surements and ensured that the nal oxygen content in the respirom-
eters was 1243.4 mm Hg, based on 90 individual measurements.
Oxygen content of the water was measured with a Strathkelvin oxy-
gen meter (Model 791, North Lanarkshire, Scotland) and Radiometer
polarographic oxygen probe (Model 1302, Brnshj, Denmark). The
ammonia excretion rate was determined as the difference in the sea
water ammonia concentration before and after 1 h in a sealed contain-
er, corrected for time and animal mass (Vosloo and Vosloo, 2010). The
ammonia content of triplicate water samples was analysed for ammo-
nia using a modied phenol-hypochlorite method (Bayne et al., 1985).
After colour development, the absorbance of the ammonia standards
and water samples was measured at 640 nm (Nanocolor 300D,
Macherey-Nagel, Duren, Germany) and concentrations derived from
the standard curve. Ammonia excretion rates were calculated as mol
nitrogen from ammonia (NH
4
N) excreted per standard body mass
(kg) and unit time (min). The ratio of atomic equivalents of oxygencon-
sumed and nitrogen excreted, or oxygen:nitrogen (O:N) ratio, was cal-
culated according to Bayne et al. (1985) from the mean of the oxygen
consumption rate and the mean of the ammonia excretion rate, as the
oxygen consumption and ammonia excretionrate data were not paired.
This did not allowfor the statistical analysis of the effect of oxygen onO:
N ratio, but the trends observed are quite clear.
Proteins were extracted from the gill and muscle tissues using the
protocols of Regoli and Principato (1995) and Drew et al. (2001)
Table 1
The different oxygen exposures (means.e.m.) for juvenile and adult abalone for the
experimental period of one month. Percentage saturation was calculated according to
Bayne et al. (1985).
Oxygen level Dissolved oxygen
(mg L
1
)
% saturation
Juveniles Hypoxia 6.400.07 82.60
Normoxia 7.700.01 97.79
Hyperoxia 9.590.09 120.25
Adults Hypoxia 6.740.002 84.52
Normoxia 7.370.002 92.43
Hyperoxia 8.950.011 112.24
193 A. Vosloo et al. / Comparative Biochemistry and Physiology, Part A 164 (2013) 192199
respectively to accommodate for the toughness of the muscle tissue.
Protein concentration was determined with the BCA Protein Assay
Kit (Pierce Biosciences) and a PowerWave XS microwell plate reader
(BioTek, Winooski, VT, USA). Haemolymph glucose concentrations
were calculated colorimetrically with a glucose assay procedure kit
(Megazyme Inc.). Haemolymph haemocyanin concentrations were
measured as described by Behrens et al. (2002), using a practical
extinction coefcient of 11.42 mmol
1
cm
1
.
2.5. Anti-oxidant enzyme activity
Anti-oxidant enzyme activity was measured using gill protein
extracts. Superoxide dismutase (SOD; EC 1.15.1.1) activity (rate %
mg prot
1
) was measured with a SOD kit (Fluka). Glutathione peroxi-
dase (GPx; EC 1.11.1.9) activity (mmol min
1
mg prot
1
) was mea-
sured with a glutathione peroxidase cellular activity kit (Sigma).
Catalase (CAT; EC 1.11.1.6) activity (nmol min
1
mg prot
1
) was
measured with a Catalase kit (Sigma).
2.6. Comet assay
The Comet assay was performed on haemocytes collected from
the pedal sinus. High melting point agarose (HMPA)-covered slides
were prepared as previously described (Vosloo et al., 2008). The
haemolymph was mixed with low melting point agarose adjusted
for marine molluscs with Kenny's salt solution (KLMA) (Lee, 2005)
and plated on the HMPA-covered slides. Slides were transferred to a
lysis buffer to ensure perforation of the nuclear envelope, for a mini-
mum of 2 days. The slides were subsequently electrophoresed (12 V,
600 mA, 40 min) to induce the migration DNA, after which slides
were stained with ethidium bromide to visualize DNA (Vosloo et al.,
2008). Under uorescent microscopy (Nikon Eclipse E400, Tokyo,
Japan) digital images of 50 comets per window were captured with
a Nikon E5400 camera. Each of the 100 comets per animal was
analysed individually (Casp 1.2 software, SourceForge, 2006) for
the following assessments of DNA fragmentation: (1) the percentage
of DNA in the tail of the comet, and (2) Olive Tail Moment (OTM).
2.7. Heat shock protein 70 (Hsp 70)
After protein extraction, accumulation of heat shock protein 70 in
muscle tissue was studied with Western blot immunodetection. Equal
amounts of protein (150 g) of each sample was added to SDS-PAGE
sample buffer, boiled for 5 min at 95 C and briey cooled on ice be-
fore use. The protein samples as well as a reference tissue extract
were resolved on pre-cast discontinuous 10% SDS-PAGE gel (Bio-Rad)
with denaturing electrophoresis. Resolved proteins were transferred
to Hybond C-extra nitrocellulose membrane (AEC Amersham) with
a dry blotter (Bio Rad, Hercules, CA, USA) for 45 min at 100 V with
cooling. Blocking and immunodetection of the resolved proteins were
performed according to AEC Amersham manual. ImmunStar HRP
Substrate solution (Bio-Rad) and a Molecular Imager VersaDoc
(Bio-Rad) were used for visualising and capturing digital images of de-
veloped membranes. Samples were normalised against -Tubulin. Im-
ages of membranes were digitally analysed with Image J 1.40 g
(National Institute of Health). Loading differences between membranes
were corrected with a standard sample on each membrane (Vosloo and
Vosloo, 2010).
2.8. Statistical analysis
Data were analysed with Prism 5 (GraphPad, USA). Data were
tested for outliers with Grubb's test with Pb0.05 and Gaussian distri-
bution was tested with the KolmogorovSmirnov test. Data were
then analysed with a two-way ANOVA, using size (juvenile/adult)
and oxygen (hypoxia/normoxia/hyperoxia) as factors, and signicant
differences were determined using the Bonferroni post hoc test. Sig-
nicant differences were assumed when Pb0.05. In order to further
analyze the nature of the DNA damage as assessed by the Comet
assay, linear regression analysis of Olive tail moment against % tail
DNA was carried out. As the regression lines were not signicantly
different between the different treatments within each size class,
the Comet data for all adults were pooled, and compared, by regres-
sion and correlation analysis, against the pooled Comet data for all
juveniles.
3. Results
Two-way analysis of variance indicated that ve of the measured
variables, viz. oxygen consumption rate, haemocyanin concentration,
SOD activity, CAT activity and hsp 70 protein levels, were affected by
life stage (juvenile vs. adult), both indicators of DNA damage (olive
tail moment and %tail DNA) were affected by bothlife stage and oxygen
concentration (hypoxia, normoxia, hyperoxia) and only two variables,
GPx activity and ammonia excretion rate, responded signicantly to
oxygen concentration.
3.1. Physiological parameters
Oxygen consumption rates were signicantly affected by life stage
(DF=1, F=30.06, Pb0.001), but not by oxygen concentration. The ox-
ygen consumption rate of the juvenile abalone exposed to hyperoxia
was signicantly higher (Pb0.001) compared to adult abalone exposed
to hyperoxia (Fig. 1A). Adult abalone exposed to hyperoxia had a higher
(Pb0.001) oxygen consumption rate compared to adults at hypoxic and
normoxic levels. As expected from allometric scaling laws, the oxygen
consumption rate of the juveniles was signicantly higher (Pb0.001)
compared to the adults at the normoxic and hypoxic oxygen levels,
but this was not valid under hyperoxic conditions.
Ammonia excretion rate was affected only by oxygen (DF=2, F=
4.218, P=0.025). Juvenile and adult abalone had similar ammonia
excretion rates (Fig. 1B) at normoxia, but exhibited different responses
to increased and decreased oxygen: ammonia excretion rate increased
(Pb0.05) in hyperoxic adults, but increased in hypoxic juveniles
(Pb0.05). No signicant changes or trends were observed in
haemolymph glucose concentrations (Table 2). Haemocyanin concentra-
tions were affected by life stage (DF=1, F=283.7, Pb0.001), with adult
abalone having signicantly higher (Pb0.001) haemocyanin concentra-
tions compared to juvenile abalone at the corresponding oxygen levels
(Table 2).
3.2. Anti-oxidant enzyme activity
SOD and CAT enzyme activities were affected by life stage (DF=1,
F=81.07 compared to Pb0.001; DF=1, F=8.997, P=0.0045 respec-
tively), whilst GPx activity responded signicantly to oxygen concentra-
tion (DF=2, F=5.894, P=0.0057). The SOD activity of the juveniles
(Table 2) was signicantly higher compared to the adults at the corre-
sponding oxygen levels (Pb0.001). GPx activity (Table 2) of the juveniles
at low oxygen was signicantly lower (Pb0.05) compared to the juve-
niles exposed to high oxygen levels. Although the trend is that SOD and
CAT activities are higher in adults than in juveniles, the signicance is
obscured by the variability in the data.
3.3. DNA damage
Both measures of DNA damage, % tail DNA and Olive tail moment,
were affected signicantly by life stage (DF=1, F=271.5, Pb0.001 and
DF=1, F=267.6, Pb0.001 respectively) and oxygen concentration
(DF=2, F=12.56, Pb0.001 andDF=2, F=12.04, Pb0.001 respectively).
Juveniles consistently had signicantly lower DNA damage in their
haemocytes thanadults (Pb0.001), but their DNAdamage didnot change
194 A. Vosloo et al. / Comparative Biochemistry and Physiology, Part A 164 (2013) 192199
in response to oxygen concentration (Fig. 1C, Table 2). Both lowand high
oxygen caused signicantly increased DNA damage in adults, when com-
pared to the normoxic group, as measured by % tail DNA and OTM
(Pb0.01). For ease of comparison, the results for DNAdamage inlarge an-
imals are reproduced from Vosloo et al. (2008), with permission of
Medimond Publishers.
3.4. Hsp 70
The hsp 70 response was signicantly affected by life stage (DF=
1, F=31.75, Pb0.001), with the relative intensity of hsp 70/-Tubulin
ratio (Fig. 2) of the adults exposed to normoxia and hyperoxia being
signicantly higher compared to the Hsp 70/-Tubulin ratio of the ju-
veniles at the corresponding oxygen levels (Pb0.01 and Pb0.001 re-
spectively). The adults exposed to hyperoxia had a signicantly
higher Hsp 70/-Tubulin ratio than the adults exposed to hypoxia
(Fig. 2B).
4. Discussion
4.1. MO
2
The resting oxygen consumption rate of normoxic adult H. midae
was similar to previously reported by Vosloo and Vosloo (2010) for
adults acclimated to 16 C. As expected, the mass specic oxygen con-
sumption rate of normoxic juvenile and adult abalone scaled to body
mass, with a calculated mass exponent of 0.22.
Like most aquatic invertebrates (Grieshaber et al., 1994), and spe-
cically H. laevigata (Harris et al., 1999), both juvenile and adult aba-
lone were able to regulate their oxygen consumption rate after
long-term exposure to low oxygen levels. The apparent insensitivity
of juvenile H. midae to moderate changes in ambient oxygen was
also observed in juvenile H. laevigata between 81 and 117% of oxygen
saturation (Harris et al., 1999).
Under more extreme hypoxia (~60% saturation), decreased oxy-
gen consumption rates have been correlated with histopathological
changes, e.g. necrosis and changes in mucus cell structure, in gills of
juvenile H. laevigata (Harris et al., 1998). As demonstrated in sh,
these histopathological changes may result in impeded oxygen diffu-
sion across gills (Van Heerden et al., 2004). As decreased oxygen con-
sumption rates were not observed in either juvenile or adult abalone
after long-term exposure to hypoxia, it is likely that the moderate
hypoxia employed in this study did not induce histopathological gill
damage or, if damage occurred, it did not limit gas exchange.
In the short-term, large H. midae conform MO
2
to low oxygen (79.8
86.7% of control MO
2
) and the regulated increase in MO
2
to pre-
exposure levels is initiated at about day four of exposure to ~80% oxygen
saturation (Vosloo et al., 2009). The physiological mechanisms that en-
able abalone to increase aerobic scope have been studied in H. iris (Ragg
and Taylor, 2006a,b), in which the maintenance of oxygen uptake at am-
bient oxygen levels above the P
crit
of 45 mm Hg is facilitated by (a) en-
dogenous ventilation by cilia on the gill lamellae, (b) enhanced water
owover the shell and (c) increased branchial blood owto enhance ox-
ygen uptake. Interestingly, virtually all oxygen uptake can be ascribed to
branchial uptake, with about 14% of the normoxic MO
2
being supplied by
the foot/epipodium in large H. iris (Taylor and Ragg, 2005). Oxygen up-
take in H. iris can be enhanced by the recruitment of the usually
poorly-perfused left gill. The resulting increased haemolymph ow and
level of oxygenation ensures that the aerobic scope of abalone can be
met (Ragg and Taylor, 2006a). The inability of adult H. midae to regulate
their resting oxygenconsumptionrate at increasing oxygenlevels is of in-
terest. Importantly, a hyperoxia-inducedincrease inoxygenconsumption
may actually result in impeded growth (Harris et al., 2005). There is thus
a need to understand howuctuations in O
2
supply will affect organismal
O
2
demand and utilization.
4.2. Ammonia excretion rate
During stressful conditions, proteins may be catabolised, releasing
CO
2
and NH
4
+
as end products (Reddy-Lopata et al., 2006; Lurman et
al., 2007b) and resulting in an increased ammonia excretion rate. This
was observed in juveniles exposed to low oxygen and adults exposed
to high oxygen. Similarly, adult craysh, Pacifastacus leniusculus, also
showed an increase in ammonia excretion rate when exposed to high
oxygen levels (Prtner et al., 2007). The increased protein catabolism
of juveniles exposed to low oxygen levels and adults exposed to high
oxygen levels is supported by the lower O:N ratio (Table 2). In zebra
mussel, an O:N ratio between 3 and 16 is indicative of exclusively
protein-based metabolism, a ratio between 50 and 60 indicates
equal utilisation of proteins and carbohydrates or lipids and an O:N
ratio above 60 indicates exclusively carbohydrate and/or lipid-based
metabolism (Quigley et al., 1997). Regardless of the changes in pro-
tein breakdown, there were no changes in the muscle or gill tissue
Fig. 1. Oxygen consumption rate (A), ammonia excretion rate (B) and (C) % tail DNA
(mean+s.e.m., n=6) of juvenile (black bars) and adult (clear bars) abalone exposed
to three oxygen levels. Dissimilar letters indicate signicance (Pb0.05).
195 A. Vosloo et al. / Comparative Biochemistry and Physiology, Part A 164 (2013) 192199
protein concentrations (Table 2) indicating that protein turnover was
at a steady, although increased, rate.
Our results conrmthe observation that haemocyanin concentrations
in abalone are highly variable (Pilson, 1965). Increased [Hc] has also been
observed as a response to hypoxic stress in blue crabs, Callinectes sapidus
(Defur et al., 1990) and Dungeness crabs, Cancer magister (Heise et al.,
2007). A putative role for Hypoxia Inducible Factor-1 in the regulation
of crustacean haemocyanin concentrations has been inferred from the
presence of possible hypoxia response elements in the promoter regions
of C. magister Hc subunit genes (Heise et al., 2007) and the discovery of
HIF-1 homologues in several crustaceans (Li and Brouwer, 2007;
Melzner et al., 2007b). The upregulationof Hc under hyperoxic conditions
can also be explained by a HIF-mediated transcriptional activation, as mi-
tochondrial ROS have been shown to stabilize HIF-1 and consequently
activate HIF-dependent downstream genes under non-hypoxic condi-
tions (Patten et al., 2010).
It is difcult to assess the functional importance of the increased
[Hc] in juveniles in response to hypoxic and hyperoxic conditions,
as the abalone oxygen transport system is geared toward storage,
not delivery (Wells et al., 1998). This is a result of the poor perfusion
of the large foot muscle (Jorgensen et al., 1984) and the reverse Bohr
shift. The magnitude of the reverse Bohr shift ranges between 0.50
and 0.57 in temperate abalone (H. iris and H. australis) (Wells et al.,
1998; Behrens et al., 2002) and 0.64 in the tropical H. asinina
(Baldwin et al., 2007), and is temperature dependent (Wells et al.,
1998). The functional signicance of the reverse Bohr shift under
hypoxic conditions is yet to be satisfactorily analysed (Behrens et
al., 2002).
4.3. Anti-oxidant enzyme activity
The ability of animals to increase antioxidant enzyme activity is an
indication of its capacity to tolerate hypoxia/anoxia (Gorr et al., 2010).
Hypoxic/anoxic tolerant animals have high antioxidant capacity in
their tissues. Our results show that juvenile abalone is more resistant
to oxidative stress because they have higher levels of antioxidant en-
zymes, specically SOD, compared to adults. Several authors have
found that hypoxic/anoxic tolerant animals have increased levels of the
antioxidant enzymes SOD and CAT (Hermes-Lima and Storey, 1993;
Willmore and Storey, 1997a,b; Hermes-Lima and Zenteno-Sav n, 2002).
This has also been demonstrated for molluscs (Pannunzio and Storey,
1998; Gorr et al., 2010). The high anti-oxidant enzyme activity serves to
prepare the animals for potential oxidative stress upon re-oxygenation
(Hermes-Lima and Zenteno-Sav n, 2002). As with other stress responses,
this response is energetically costly (Gorr et al., 2010).
The anti-oxidant enzyme, SOD, converts free radicals to H
2
O
2
,
where-after GPx and CAT reduce H
2
O
2
to H
2
O and O
2
(Mats and
Snchez-Jimnez, 1999; Kooter, 2004). When the H
2
O
2
levels are
lower than 10 mol L
1
, GPx is responsible for the removal of 8085%
of the H
2
O
2
, whereas CAT removes H
2
O
2
when concentrations exceed
10 mol L
1
(Hashida et al., 2002).
Fig. 2. A composite image of pooled samples in duplicate to illustrate the intensity of the
Hsp 70 and -Tubulinbands inmuscle tissue for juvenile (A) and adult (B) abalone during
hypoxia, normoxia and hyperoxia. Std = standard sample. (C) Hsp 70/-Tubulin ratios
(means.e.m., n=6) of juvenile (black bars) and adult (clear bars) abalone exposed to
hypoxia, normoxia and hyperoxia. Dissimilar letters indicate signicance (Pb0.05).
Table 2
Physiological and biochemical effects of long term exposure to low and high oxygen on H. midae.
Juveniles Adults
Hypoxia Normoxia (control) Hyperoxia Hypoxia Normoxia (control) Hyperoxia
O:N 18.75 179.22 173.08 138.17 114.8 62.26
Muscle protein
(mg g
1
)
97.0098.18 19.148.6 7.813.91 938.76 102.16.0 104.605.55
Gill protein
(mg g
1
)
142.6314.38 121.6118.2 129.2513.68 128.8014.85 112.6016.75 132.7025.06
Glucose
(mmol L
1
)
0.300.52 0.420.08 0.660.14
\
0.590.24 0.230.02 0.460.15
Haemocyanin
(mol functional Hc units L
1
)
14.082.07
abc
11.320.73
acd
16.251.32
abf
99.677.45
def
115.6014.13
be
91.5311.75
cde
SOD
(rate % mg prot
1
)
1.990.22
abc
2.180.39
acd
1.710.14
abf
0.080.03
def
0.150.03
be
0.180.03
cde
GPx
mol min
1
mg prot
1
0.030.01
b
0.1 0.02
ab
0.150.04
a
0.020.01
ab
0.160.06
ab
0.170.08
ab
CAT
(mmol min
1
mg prot
1
)
30.3111.54 23.196.13 17.964.33 8.432.72 5.290.85 3.260.89
OTM
(arbitrary units)
6.590.76
acd
5.750.71
abd
6.340.54
abc
30.993.60
be
18.475.02
c
30.495.94
de
Values given as means.e.m. of n=410, except for oxygen:nitrogen (O:N) ratio, which was calculated from the mean oxygen consumption rate and the mean ammonia excretion
rate. Different letters indicate signicance within and between size classes for each parameter. Olive Tail Moment (OTM) data for adults were reproduced from Vosloo et al. (2008),
with permission from Medimond Publishers. SOD = superoxide dismutase; GPx = glutathione peroxidase; CAT = catalase.
196 A. Vosloo et al. / Comparative Biochemistry and Physiology, Part A 164 (2013) 192199
In adult abalone, the non-signicant trend of increased SOD activ-
ity with increased oxygen levels, indicates that more oxyradicals
were present (Lurman et al., 2007a). However, GPx and CAT activities
did not increase to remove the high levels of H
2
O
2
resulting from the
slightly elevated SOD activity. The lack of change in GPx and CAT ac-
tivities may result from their inhibition by accumulated oxyradicals
(Melzner et al., 2007a). The fact that increased DNA damage was in-
deed observed in the haemocytes of adult abalone under hypoxic
and hyperoxic conditions (Fig. 1C) leads to the conclusion that the
anti-oxidant capacity has been exceeded. Although not measured, it
is likely that oxidative damage to lipids and proteins (Lurman et al.,
2007a) will also manifest in adult abalone under these conditions.
4.4. DNA damage
The DNA damage theory of ageing (Freitas and de Magalhaes,
2011) proposes that DNA damage accumulates during the lifespan
of organisms as a result of the gradual breakdown in DNA repair
mechanisms. Consequently older organisms have higher baseline
levels of DNA damage than younger ones. This trend is also observed
when comparing juvenile and adult abalone under normoxic condi-
tions (Fig. 1C).
Juvenile and adult abalone responded differently to changes in en-
vironmental oxygen. Juveniles did not exhibit any changes in DNA
damage in response to changes in environmental oxygen. Because ob-
served DNA integrity is a result of the dynamic processes of damage
and repair, juvenile abalone suffered less damage due to intrinsically
better removal of ROS by scavenger molecules (Lurman et al., 2007a),
improved ability to recruit anti-oxidant enzymes like GPx (Table 2)
(Gorr et al., 2010), higher potential to repair DNA damage (Pacici
and Davies, 1991) or a combination of the above. In contrast, both
the percentage tail DNA (Fig. 1C) and OTM (Table 2) for the adults
were higher under hypoxic and hyperoxic conditions. The inability
of the adults to prevent and/or repair DNA damage can result from
a decreased ability to repair DNA damage with an increase in age
(Pacici and Davies, 1991; Freitas and de Magalhaes, 2011).
The correlation analysis (Fig. 3) indicates that juveniles, generally,
had large amounts of DNA in the tail of the comets (as measured by %
tail DNA) and shorter tail lengths (as measured by the Olive tail mo-
ment). This is indicative of single strand breaks (SSBs) in the DNA, as
the long strands of DNA cannot move very far through the agarose gel
during the SCGE electrophoresis (Fornace et al., 1976; Klaude et al.,
1996). Repair of SSBs is relatively easy, as the unbroken strand can
be used as a template to guide correction of the damaged strand
(Kumaravel et al., 2009). In contrast, DNA damage in adult abalone
appears to be predominantly double stranded breaks (DSBs), as in-
ferred from the low percentage DNA in the tails, but longer comet
tail lengths. Double-strand breaks are particularly dangerous to the
cell as both strands are severed, making the damage harder to repair.
Such damage can lead to rearrangement in the actual genome, which
is wholly more detrimental to the animal and future generations
(Watson et al., 2007).
4.5. Hsp 70
In human cells, an increase in HSPs can cause a decrease in oxida-
tive damage (Lurman et al., 2007a). This is possible since HSPs are in-
duced as a cellular stress response in repairing damage to existing
proteins (Decker et al., 2007) and by moving proteins beyond repair
to proteosomes where the protein will be degraded through proteol-
ysis (Lurman et al., 2007a). In oysters, Crassostrea gigas, (David et al.,
2005) and clams, Donax variabilis, (Joyner-Matos et al., 2006), in-
creased hsp70 expression, and hence increased hsp 70 proteins
(Anestis et al., 2007) was observed in response to both hypoxic and
hyperoxic conditions.
Juvenile abalone had lower hsp 70/-Tubulin ratios compared to
adults under normoxia and hyperoxia (Fig. 2). It has been demon-
strated that puried hsp 70 from young rats offer more protection
to enzyme reactions proceeding under heated conditions (Shpund
and Gershon, 1997), thus it follows that that more hsp 70 would be
required to provide protection at a basal level. In addition, the ob-
served increases in oxidative DNA damage observed in adult abalone
possibly also results in oxidative protein damage, requiring a further
increase in hsp 70 proteins for either protective, repair or degradation
tagging functions.
5. Conclusion
The results clearly indicate that, physiologically and biochemical-
ly, juvenile and adult abalone reacted differently to changes in envi-
ronmental oxygen levels. The physiological adjustments allowed the
juveniles to prevent damage to DNA and, judging by the hsp 70 levels,
also to proteins. Therefore, the juveniles were more effective in their
protection against different oxygen levels above and below satura-
tion. In contrast, although adult abalone employed an array of organ-
ismal, biochemical and cellular processes, they were unable to
prevent DNA and protein damage in response to changes in environ-
mental oxygen levels. The energetic cost of these protective re-
sponses limits the scope for growth, and results in slower growth of
abalone (Vosloo et al., 2008). This implies that the animals might
not reach market size in four years and consequently cause an in-
crease in production costs. The current farm practice of tightly con-
trolling the environmental conditions for juveniles and keeping
adults under more exposed and variable grow-out conditions appears
to be in conict with our results, which show that juvenile abalone
can successfully adapt to changes in environmental oxygen and tem-
perature (D. Vosloo, A. Vosloo, unpublished). The apparent insensitiv-
ity of juvenile abalone to changes in oxygen is probably a residual
response from their exposure to naturally uctuating oxygen levels
in the diatom biolm.
The importance of understanding animal performance in aquacul-
ture systems should not be underestimated. The capacity of physio-
logical systems to cope with environmental conditions that differ to
varying degrees from the natural environment, can inform discus-
sions of potential future biological effects of global climate change.
Farmed abalone as a test species is useful, as individual animals
spend up to four years in the farm environment. However, slight
changes of the natural environmental conditions on abalone farms
exert some degree of pressure on the existing physiological machin-
ery of abalone. In addition, adult abalone used for spawning and pro-
duction of offspring are usually individuals collected from the wild
Fig. 3. Regression analysis of % tail DNA against Olive tail moment of juvenile (black cir-
cles; n=1500, slope=0.42, r
2
=0.39) and adult (clear circles; n=500, slope=0.96,
r
2
=0.90) haemocytes subjected to the Comet assay. The lines are signicantly differ-
ent (F=1856.9, Pb0.0001), and indicate that the haemocytes of juvenile abalone
have more single strand breaks in the DNA, whilst the DNA of adult haemocytes
have more double strand breaks.
197 A. Vosloo et al. / Comparative Biochemistry and Physiology, Part A 164 (2013) 192199
and are kept at regulated ambient temperature and day/night cycles;
consequently comparable generations of offspring, of a range of sizes,
exist on a particular farm. In a limited number of instances, a farm
may use F1 generation of farm-produced animals as broodstock, pro-
viding yet another interesting avenue of determining generational in-
uence of the changed farm environment, as proxy for global climate
change.
Funding
I & J Abalone Division supplied the animals and articial feed used
for experimentation, as well as the infrastructure required to hold the
animals during the exposures. Funding was made available to AV
through the Frontier Programme, project NWU2005, from which all
consumables and running costs were covered.
Acknowledgements
The authors would like to thank for I & J Abalone Division, Gansbaai
for the experimental animals and Marine & Coastal Management
(M&CM) through the Frontier Programme (NWU2005) for funding.
We would also like to thank Lize Schoonbee and Deirdre Snyman at I
& J Abalone Division and Drien Wolmarans for the technical assistance.
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