Comparative Biochemistry and Physiology, Part A 164 (2013) 192–199

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Comparative Biochemistry and Physiology, Part A

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Differential responses of juvenile and adult South African abalone (Haliotis midaeLinnaeus) to low and high oxygen levels

Andre Vosloo ⁎, Anél Laas, Dalene VoslooSchool of Life Sciences, University of KwaZulu-Natal, Private Bag X54001, Durban, 4000, South Africa

⁎ Corresponding author. Tel.: +27 312601337.E-mail address: vosloo@ukzn.ac.za (A. Vosloo).

1095-6433/$ – see front matter © 2012 Elsevier Inc. Allhttp://dx.doi.org/10.1016/j.cbpa.2012.09.002

a b s t r a c t

a r t i c l e i n f o

Article history:Received 16 May 2012Received in revised form 4 September 2012Accepted 4 September 2012Available online 10 September 2012

Keywords:Haliotis midaeAbaloneOxidative stressPhysiologyComet assayHsp 70Catalase

Marine invertebrates have evolved multiple responses to naturally variable environmental oxygen, all aimedat either maintaining cellular oxygen homeostasis or limiting cellular damage during or after hypoxic orhyperoxic 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 tolow (~83% of saturation), intermediate (~95% of saturation) and high (~115% of saturation) oxygen levels forone month. Using the Comet assay, we measured DNA damage to determine whether the observed trends inthe protective responses were sufficient to prevent oxidative damage to cells. Juveniles were unaffected bymoderately hypoxic and hyperoxic conditions. Elevated basal rates of superoxide dismutase, glutathione per-oxidase and catalase were sufficient to prevent DNA fragmentation and protein damage. Adults, with theirlower 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 hypoxicand hyperoxic conditions. The apparent insensitivity of juvenile abalone to decreased and increased oxygenmight be related to their life history and development in algal and diatom biofilms where they are exposed toextreme diurnal fluctuations in dissolved oxygen levels.

© 2012 Elsevier Inc. All rights reserved.

1. Introduction

Temperature and oxygen are the most important environmentalfactors that govern biological processes in nature. Temperature, forinstance, may directly or indirectly drive biogeographical patterns(Pörtner 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 inefficient production (growth and recruitment)at the edges of the distribution range and (b) eventual exclusion ofspecies outside specific temperature and oxygen ranges. Just as natu-ral temperature and oxygen ranges influence production on the bio-geographical scale, temperature and oxygen profiles of intensiveaquaculture systems determine production and, in this sense, finan-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 itsnatural Western and Southern distributions, Haliotis midae can be ex-posed to temporary bouts of anoxia or hypoxia resulting from thedecay 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

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would be unable to escape these conditions and consequently haveto initiate adaptive responses to the hypoxic conditions.

In the diatom biofilm 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 phasephotosynthesis (Daume, 2006; Roberts et al., 2007). Due to theirsmall size and limited mobility, larval and juvenile abalone cannot es-cape these extreme diurnal oxygen fluctuations. After growing out ofthe diatom biofilm, 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 ofoxygen utilization (e.g. biomass, stocking density) and oxygen supply(e.g. water flow rate, aeration efficiency, temperature-dependent ox-ygen solubility). Dissolved oxygen concentrations may fluctuate be-tween 8.2 and 7.2 mg O2L−1 in abalone aquaculture systems inSouth Africa, and depend on the distance from the tank inlet andtime of day (Yearsley, 2007). Water flow may also decrease or ceaseduring periods of power outages, leading to temporary decreases indissolved oxygen. Farm management interventions e.g. tank/racewaycleaning and routine weighing and redistribution of biomass, result inanimals being removed from water, spending variable amounts oftime in air, with resultant oxygen supply limitation (Vosloo et al.,2008). This periodic air exposure may limit the oxygen extraction ef-ficiency of the abalone respiratory system, as adult H. midae occursnaturally in crevices and on shallow reefs, but reach maximum

Table 1The different oxygen exposures (mean±s.e.m.) for juvenile and adult abalone for theexperimental period of one month. Percentage saturation was calculated according toBayne et al. (1985).

Oxygen level Dissolved oxygen(mg L−1)

% saturation

Juveniles Hypoxia 6.40±0.07 82.60Normoxia 7.70±0.01 97.79Hyperoxia 9.59±0.09 120.25

Adults Hypoxia 6.74±0.002 84.52Normoxia 7.37±0.002 92.43Hyperoxia 8.95±0.011 112.24

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densities permanently inundated in kelp (Ecklonia maxima) forests(Branch et al., 2008).

Although reactive oxygen species (ROS) are formed during normalmitochondrial processes, ROS levels may increase if there is a mismatchbetween oxygen supply and demand. The accurate matching of cellularoxygen 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 beadjusted by decreased reliance on a hierarchy of high-demand cellularprocesses like RNA and protein synthesis, ion transport, gluconeogene-sis and proton leak (Bishop et al., 2002; Staples and Buck, 2009). Inmammals this attenuation is highly ROS‐dependent (Brookes et al.,2004).

Biological systems have complex mechanisms to prevent ROS-induced damage (Lurman et al., 2007a). It has been suggested thatthis 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, α-tocopherol) and inducible proteins such as transferrinand 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 componentsand (c) the cell's natural repair systems can be induced to repair damagedproteins or DNA (Manduzio et al., 2005; Lurman et al., 2007a). ShouldROS levels exceed the rate of scavenging, detoxification and repair, oxida-tive stress as measured by ROS induced damage to proteins, lipids andDNA will manifest.

From the above discussions it is clear that the environmental con-ditions for post-larval, juvenile and adult abalone differ markedly interms of oxygen content, leading to the hypothesis that their re-sponses to increased or decreased ambient oxygen would differ. Theaim of the study reported here was to assess the extent to which ab-alone, with their relatively primitive respiratory system, are able todeal with moderately increased or decreased oxygen saturation. Re-sponses included organismal level responses (oxygen consumption,ammonia excretion), substrate utilization (O:N ratio, haemolymphglucose and tissue protein levels), indicators of gas exchange (D-lactate, haemocyanin concentration), antioxidant protection (SOD,GPx and CAT activity), cellular repair processes (hsp70 proteins) andROS-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; 12.2±0.27 g; 41.4±0.27 mm shell length) and adult abalone (N=382; 53.6±0.24 g;65.32±0.15 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 awater flow rate of approximately 20 L h−1 ensuring one water ex-change in the containers every hour. Water temperature was regulatedat 16 °C by a central heater/cooler and oxygen concentration was regu-lated at 7.57±0.002 mg L−1 O2 (mean±s.e.m.) by aeration. The hold-ing tanks were cleaned twice a week and animals were provided with0.015 g artificial abalone feed (Abfeed S34, Marifeed (Pty) Ltd) pergram 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 levelsfor one month (see Table 1 for details). In the interest of consistencyand 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 airwas bubbled through the water. Higher and lower than saturation oxy-gen levels weremaintained by bubbling atmospheric air supplementedwith pure oxygen and nitrogen respectively through the water. Watertemperature was regulated at 16 °C. Water temperature and oxygenlevels were measured in the morning and afternoon each day (YSI556Multiparameter System) and adjustments to the oxygen or nitrogenflow were made when necessary (Table 1). Previous studies on greenlip abalone (Haliotis laevigata) used similar oxygen concentrations(Harris et al., 1999).

2.3. Sampling

After one month of exposure, haemolymph was sampled from thepedal 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-Natalfor analysis. Haemolymph samples for the Comet assay were kept onice until analysis.

2.4. Physiological parameters

Mass specific oxygen consumption rate of individual animals wasmeasured in closed systems using sealed respirometers. Respirometervolumes were adjusted between 200 and 660 mL, and sealed timesbetween 10 and 110 min, depending on the size of the abalone. Thisapproach prevented excessive oxygen content decrease during mea-surements and ensured that the final oxygen content in the respirom-eters was 124±3.4 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 Radiometerpolarographic oxygen probe (Model 1302, Brønshøj, Denmark). Theammonia excretion rate was determined as the difference in the seawater ammonia concentration before and after 1 h in a sealed contain-er, corrected for time and animal mass (Vosloo and Vosloo, 2010). Theammonia content of triplicate water samples was analysed for ammo-nia using a modified phenol-hypochlorite method (Bayne et al., 1985).After colour development, the absorbance of the ammonia standardsand water samples was measured at 640 nm (Nanocolor® 300D,Macherey-Nagel, Duren, Germany) and concentrations derived fromthe standard curve. Ammonia excretion rates were calculated as μmolnitrogen from ammonia (NH4–N) excreted per standard body mass(kg) and unit time (min). The ratio of atomic equivalents of oxygen con-sumed and nitrogen excreted, or oxygen:nitrogen (O:N) ratio, was cal-culated according to Bayne et al. (1985) from the mean of the oxygenconsumption rate and the mean of the ammonia excretion rate, as theoxygen consumption and ammonia excretion rate datawere not paired.This did not allow for 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 theprotocols of Regoli and Principato (1995) and Drew et al. (2001)

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respectively to accommodate for the toughness of the muscle tissue.Protein concentration was determined with the BCA Protein AssayKit (Pierce Biosciences) and a PowerWave XS microwell plate reader(BioTek, Winooski, VT, USA). Haemolymph glucose concentrationswere calculated colorimetrically with a glucose assay procedure kit(Megazyme Inc.). Haemolymph haemocyanin concentrations weremeasured as described by Behrens et al. (2002), using a practicalextinction coefficient of 11.42 mmol−1 cm−1.

2.5. Anti-oxidant enzyme activity

Anti-oxidant enzyme activity was measured using gill proteinextracts. 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) wasmeasured with a Catalase kit (Sigma).

2.6. Comet assay

The Comet assay was performed on haemocytes collected fromthe pedal sinus. High melting point agarose (HMPA)-covered slideswere prepared as previously described (Vosloo et al., 2008). Thehaemolymph was mixed with low melting point agarose adjustedfor marine molluscs with Kenny's salt solution (KLMA) (Lee, 2005)and plated on the HMPA-covered slides. Slides were transferred to alysis 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 slideswere stained with ethidium bromide to visualize DNA (Vosloo et al.,2008). Under fluorescent microscopy (Nikon Eclipse E400, Tokyo,Japan) digital images of 50 comets per window were captured witha Nikon E5400 camera. Each of the 100 comets per animal wasanalysed individually (Casp 1.2 software, SourceForge®, 2006) forthe following assessments of DNA fragmentation: (1) the percentageof 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 inmuscle tissue was studied withWestern blot immunodetection. Equalamounts of protein (150 μg) of each sample was added to SDS-PAGEsample buffer, boiled for 5 min at 95 °C and briefly cooled on ice be-fore use. The protein samples as well as a reference tissue extractwere resolved on pre-cast discontinuous 10% SDS-PAGE gel (Bio-Rad)with denaturing electrophoresis. Resolved proteins were transferredto Hybond C-extra nitrocellulose membrane (AEC Amersham) witha dry blotter (Bio Rad, Hercules, CA, USA) for 45 min at 100 V withcooling. Blocking and immunodetection of the resolved proteins wereperformed according to AEC Amersham manual. ImmunStar™ HRPSubstrate 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 α-Tubulin. Im-ages of membranes were digitally analysed with Image J 1.40 g(National Institute of Health). Loading differences betweenmembraneswere corrected with a standard sample on eachmembrane (Vosloo andVosloo, 2010).

2.8. Statistical analysis

Data were analysed with Prism 5 (GraphPad, USA). Data weretested for outliers with Grubb's test with Pb0.05 and Gaussian distri-bution was tested with the Kolmogorov–Smirnov test. Data werethen analysed with a two-way ANOVA, using size (juvenile/adult)and oxygen (hypoxia/normoxia/hyperoxia) as factors, and significant

differences were determined using the Bonferroni post hoc test. Sig-nificant differences were assumed when Pb0.05. In order to furtheranalyze the nature of the DNA damage as assessed by the Cometassay, linear regression analysis of Olive tail moment against % tailDNA was carried out. As the regression lines were not significantlydifferent 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 alljuveniles.

3. Results

Two-way analysis of variance indicated that five of the measuredvariables, viz. oxygen consumption rate, haemocyanin concentration,SOD activity, CAT activity and hsp 70 protein levels, were affected bylife stage (juvenile vs. adult), both indicators of DNA damage (olivetailmoment and % tail DNA)were affected by both life stage and oxygenconcentration (hypoxia, normoxia, hyperoxia) and only two variables,GPx activity and ammonia excretion rate, responded significantly tooxygen concentration.

3.1. Physiological parameters

Oxygen consumption rates were significantly 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 hyperoxiawas significantly higher (Pb0.001) compared to adult abalone exposedto hyperoxia (Fig. 1A). Adult abalone exposed to hyperoxia had a higher(Pb0.001) oxygen consumption rate compared to adults at hypoxic andnormoxic levels. As expected from allometric scaling laws, the oxygenconsumption rate of the juveniles was significantly 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 ammoniaexcretion rates (Fig. 1B) at normoxia, but exhibited different responsesto increased and decreased oxygen: ammonia excretion rate increased(Pb0.05) in hyperoxic adults, but increased in hypoxic juveniles(Pb0.05). No significant changes or trends were observed inhaemolymph glucose concentrations (Table 2). Haemocyanin concentra-tions were affected by life stage (DF=1, F=283.7, Pb0.001), with adultabalone having significantly 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 significantly to oxygen concentra-tion (DF=2, F=5.894, P=0.0057). The SOD activity of the juveniles(Table 2) was significantly higher compared to the adults at the corre-sponding oxygen levels (Pb0.001). GPx activity (Table 2) of the juvenilesat low oxygen was significantly lower (Pb0.05) compared to the juve-niles exposed to high oxygen levels. Although the trend is that SOD andCAT activities are higher in adults than in juveniles, the significance isobscured by the variability in the data.

3.3. DNA damage

Both measures of DNA damage, % tail DNA and Olive tail moment,were affected significantly by life stage (DF=1, F=271.5, Pb0.001 andDF=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 significantly lower DNA damage in theirhaemocytes than adults (Pb0.001), but their DNAdamagedid not change

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in response to oxygen concentration (Fig. 1C, Table 2). Both low and highoxygen caused significantly 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 in large an-imals are reproduced from Vosloo et al. (2008), with permission ofMedimond Publishers.

3.4. Hsp 70

The hsp 70 response was significantly affected by life stage (DF=1, F=31.75, Pb0.001), with the relative intensity of hsp 70/α-Tubulinratio (Fig. 2) of the adults exposed to normoxia and hyperoxia beingsignificantly higher compared to the Hsp 70/α-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 significantlyhigher Hsp 70/α-Tubulin ratio than the adults exposed to hypoxia(Fig. 2B).

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 exposedto three oxygen levels. Dissimilar letters indicate significance (Pb0.05).

4. Discussion

4.1. MO2

The resting oxygen consumption rate of normoxic adult H. midaewas similar to previously reported by Vosloo and Vosloo (2010) foradults acclimated to 16 °C. As expected, the mass specific oxygen con-sumption rate of normoxic juvenile and adult abalone scaled to bodymass, with a calculated mass exponent of −0.22.

Like most aquatic invertebrates (Grieshaber et al., 1994), and spe-cifically H. laevigata (Harris et al., 1999), both juvenile and adult aba-lone were able to regulate their oxygen consumption rate afterlong-term exposure to low oxygen levels. The apparent insensitivityof juvenile H. midae to moderate changes in ambient oxygen wasalso observed in juvenile H. laevigata between 81 and 117% of oxygensaturation (Harris et al., 1999).

Under more extreme hypoxia (~60% saturation), decreased oxy-gen consumption rates have been correlated with histopathologicalchanges, e.g. necrosis and changes in mucus cell structure, in gills ofjuvenile H. laevigata (Harris et al., 1998). As demonstrated in fish,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 abaloneafter long-term exposure to hypoxia, it is likely that the moderatehypoxia employed in this study did not induce histopathological gilldamage or, if damage occurred, it did not limit gas exchange.

In the short-term, large H. midae conform MO2 to low oxygen (79.8–86.7% of control MO2) and the regulated increase in MO2 to pre-exposure levels is initiated at about day four of exposure to ~80% oxygensaturation (Vosloo et al., 2009). The physiological mechanisms that en-able abalone to increase aerobic scope have been studied in H. iris (Raggand Taylor, 2006a,b), in which the maintenance of oxygen uptake at am-bient oxygen levels above the Pcrit of 45 mm Hg is facilitated by (a) en-dogenous ventilation by cilia on the gill lamellae, (b) enhanced waterflow over the shell and (c) increased branchial blood flow to enhance ox-ygen uptake. Interestingly, virtually all oxygen uptake can be ascribed tobranchial uptake, with about 14% of the normoxic MO2 being supplied bythe foot/epipodium in large H. iris (Taylor and Ragg, 2005). Oxygen up-take in H. iris can be enhanced by the recruitment of the usuallypoorly-perfused left gill. The resulting increased haemolymph flow andlevel of oxygenation ensures that the aerobic scope of abalone can bemet (Ragg and Taylor, 2006a). The inability of adult H. midae to regulatetheir resting oxygen consumption rate at increasing oxygen levels is of in-terest. Importantly, a hyperoxia-induced increase in oxygen consumptionmay actually result in impeded growth (Harris et al., 2005). There is thusa need to understand how fluctuations in O2 supplywill affect organismalO2 demand and utilization.

4.2. Ammonia excretion rate

During stressful conditions, proteins may be catabolised, releasingCO2 and NH4

+ as end products (Reddy-Lopata et al., 2006; Lurman etal., 2007b) and resulting in an increased ammonia excretion rate. Thiswas observed in juveniles exposed to low oxygen and adults exposedto high oxygen. Similarly, adult crayfish, Pacifastacus leniusculus, alsoshowed an increase in ammonia excretion rate when exposed to highoxygen levels (Pörtner et al., 2007). The increased protein catabolismof juveniles exposed to low oxygen levels and adults exposed to highoxygen levels is supported by the lower O:N ratio (Table 2). In zebramussel, an O:N ratio between 3 and 16 is indicative of exclusivelyprotein-based metabolism, a ratio between 50 and 60 indicatesequal utilisation of proteins and carbohydrates or lipids and an O:Nratio above 60 indicates exclusively carbohydrate and/or lipid-basedmetabolism (Quigley et al., 1997). Regardless of the changes in pro-tein breakdown, there were no changes in the muscle or gill tissue

Table 2Physiological 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 62.26Muscle protein(mg g−1)

97.00±98.18 19.14±8.6 7.81±3.91 93±8.76 102.1±6.0 104.60±5.55

Gill protein(mg g−1)

142.63±14.38 121.61±18.2 129.25±13.68 128.80±14.85 112.60±16.75 132.70±25.06

Glucose(mmol L−1)

0.30±0.52 0.42±0.08 0.66±0.14\ 0.59±0.24 0.23±0.02 0.46±0.15

Haemocyanin(μmol functional Hc units L−1)

14.08±2.07abc 11.32±0.73acd 16.25±1.32abf 99.67±7.45def 115.60±14.13be 91.53±11.75cde

SOD(rate % mg prot−1)

1.99±0.22abc 2.18±0.39acd 1.71±0.14abf 0.08±0.03def 0.15±0.03be 0.18±0.03cde

GPxμmol min−1 mg prot−1

0.03±0.01b 0.1 ±0.02ab 0.15±0.04a 0.02±0.01ab 0.16±0.06ab 0.17±0.08ab

CAT(mmol min−1 mg prot−1)

30.31±11.54 23.19±6.13 17.96±4.33 8.43±2.72 5.29±0.85 3.26±0.89

OTM(arbitrary units)

6.59±0.76acd 5.75±0.71abd 6.34±0.54abc 30.99±3.60be 18.47±5.02c 30.49±5.94de

Values given as mean±s.e.m. of n=4–10, except for oxygen:nitrogen (O:N) ratio, which was calculated from the mean oxygen consumption rate and the mean ammonia excretionrate. Different letters indicate significance 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.

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protein concentrations (Table 2) indicating that protein turnover wasat a steady, although increased, rate.

Our results confirm the observation that haemocyanin concentrationsin abalone are highly variable (Pilson, 1965). Increased [Hc] has also beenobserved 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 regulationof crustacean haemocyanin concentrations has been inferred from thepresence of possible hypoxia response elements in the promoter regions

Fig. 2. A composite image of pooled samples in duplicate to illustrate the intensity of theHsp 70 andα-Tubulin bands inmuscle tissue for juvenile (A) and adult (B) abalone duringhypoxia, normoxia and hyperoxia. Std = standard sample. (C) Hsp 70/α-Tubulin ratios(mean±s.e.m., n=6) of juvenile (black bars) and adult (clear bars) abalone exposed tohypoxia, normoxia and hyperoxia. Dissimilar letters indicate significance (Pb0.05).

of C. magister Hc subunit genes (Heise et al., 2007) and the discovery ofHIF-1α homologues in several crustaceans (Li and Brouwer, 2007;Melzner et al., 2007b). The upregulation ofHcunder hyperoxic conditionscan also be explained by a HIF-mediated transcriptional activation, asmi-tochondrial ROS have been shown to stabilize HIF-1α and consequentlyactivate HIF-dependent downstream genes under non-hypoxic condi-tions (Patten et al., 2010).

It is difficult 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 perfusionof the large foot muscle (Jorgensen et al., 1984) and the reverse Bohrshift. The magnitude of the reverse Bohr shift ranges between 0.50and 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 significance of the reverse Bohr shift underhypoxic conditions is yet to be satisfactorily analysed (Behrens etal., 2002).

4.3. Anti-oxidant enzyme activity

The ability of animals to increase antioxidant enzyme activity is anindication of its capacity to tolerate hypoxia/anoxia (Gorr et al., 2010).Hypoxic/anoxic tolerant animals have high antioxidant capacity intheir tissues. Our results show that juvenile abalone is more resistantto oxidative stress because they have higher levels of antioxidant en-zymes, specifically SOD, compared to adults. Several authors havefound that hypoxic/anoxic tolerant animals have increased levels of theantioxidant enzymes SOD and CAT (Hermes-Lima and Storey, 1993;Willmore and Storey, 1997a,b; Hermes-Lima and Zenteno-Savı́n, 2002).This has also been demonstrated for molluscs (Pannunzio and Storey,1998; Gorr et al., 2010). The high anti-oxidant enzyme activity serves toprepare the animals for potential oxidative stress upon re-oxygenation(Hermes-Lima and Zenteno-Savı́n, 2002). As with other stress responses,this response is energetically costly (Gorr et al., 2010).

The anti-oxidant enzyme, SOD, converts free radicals to H2O2,where-after GPx and CAT reduce H2O2 to H2O and O2 (Matés andSánchez-Jiménez, 1999; Kooter, 2004). When the H2O2 levels arelower than 10 μmol L−1, GPx is responsible for the removal of 80–85%of the H2O2, whereas CAT removes H2O2 when concentrations exceed10 μmol L−1 (Hashida et al., 2002).

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In adult abalone, the non-significant trend of increased SOD activ-ity with increased oxygen levels, indicates that more oxyradicalswere present (Lurman et al., 2007a). However, GPx and CAT activitiesdid not increase to remove the high levels of H2O2 resulting from theslightly 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 hypoxicand hyperoxic conditions (Fig. 1C) leads to the conclusion that theanti-oxidant capacity has been exceeded. Although not measured, itis 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 lifespanof organisms as a result of the gradual breakdown in DNA repairmechanisms. Consequently older organisms have higher baselinelevels of DNA damage than younger ones. This trend is also observedwhen 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 DNAdamage in response to changes in environmental oxygen. Because ob-served DNA integrity is a result of the dynamic processes of damageand repair, juvenile abalone suffered less damage due to intrinsicallybetter 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 (Pacificiand Davies, 1991) or a combination of the above. In contrast, boththe percentage tail DNA (Fig. 1C) and OTM (Table 2) for the adultswere higher under hypoxic and hyperoxic conditions. The inabilityof the adults to prevent and/or repair DNA damage can result froma decreased ability to repair DNA damage with an increase in age(Pacifici 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, asthe long strands of DNA cannot move very far through the agarose gelduring the SCGE electrophoresis (Fornace et al., 1976; Klaude et al.,1996). Repair of SSBs is relatively easy, as the unbroken strand canbe used as a template to guide correction of the damaged strand(Kumaravel et al., 2009). In contrast, DNA damage in adult abalone

Fig. 3. Regression analysis of % tail DNA against Olive tail moment of juvenile (black cir-cles; n=1500, slope=0.42, r2=0.39) and adult (clear circles; n=500, slope=0.96,r2=0.90) haemocytes subjected to the Comet assay. The lines are significantly differ-ent (F=1856.9, Pb0.0001), and indicate that the haemocytes of juvenile abalonehave more single strand breaks in the DNA, whilst the DNA of adult haemocyteshave more double strand breaks.

appears to be predominantly double stranded breaks (DSBs), as in-ferred from the low percentage DNA in the tails, but longer comettail lengths. Double-strand breaks are particularly dangerous to thecell as both strands are severed, making the damage harder to repair.Such damage can lead to rearrangement in the actual genome, whichis 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 existingproteins (Decker et al., 2007) and by moving proteins beyond repairto 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 andhyperoxic conditions.

Juvenile abalone had lower hsp 70/α-Tubulin ratios compared toadults under normoxia and hyperoxia (Fig. 2). It has been demon-strated that purified hsp 70 from young rats offer more protectionto enzyme reactions proceeding under heated conditions (Shpundand Gershon, 1997), thus it follows that that more hsp 70 would berequired to provide protection at a basal level. In addition, the ob-served increases in oxidative DNA damage observed in adult abalonepossibly also results in oxidative protein damage, requiring a furtherincrease in hsp 70 proteins for either protective, repair or degradationtagging 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 thejuveniles to prevent damage to DNA and, judging by the hsp 70 levels,also to proteins. Therefore, the juveniles were more effective in theirprotection 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 toprevent 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 ofabalone (Vosloo et al., 2008). This implies that the animals mightnot 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 keepingadults under more exposed and variable grow-out conditions appearsto be in conflict with our results, which show that juvenile abalonecan 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 residualresponse from their exposure to naturally fluctuating oxygen levelsin the diatom biofilm.

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 tovarying 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 animalsspend up to four years in the farm environment. However, slightchanges of the natural environmental conditions on abalone farmsexert 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

198 A. Vosloo et al. / Comparative Biochemistry and Physiology, Part A 164 (2013) 192–199

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 farmmay use F1 generation of farm-produced animals as broodstock, pro-viding yet another interesting avenue of determining generational in-fluence of the changed farm environment, as proxy for global climatechange.

Funding

I & J Abalone Division supplied the animals and artificial feed usedfor experimentation, as well as the infrastructure required to hold theanimals during the exposures. Funding was made available to AVthrough the Frontier Programme, project NWU2005, from which allconsumables and running costs were covered.

Acknowledgements

The authors would like to thank for I & J Abalone Division, Gansbaaifor 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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