Ellen Weihe

Michael Kriews

Doris Abele
email: doris.abele@awi.de

Differences in heavy metal concentrations and in the response of the antioxidant system to hypoxia and air exposure in the Antarctic limpet Nacella concinna

Differences in heavy metal concentrations and in the response of the antioxidant system to hypoxia and air exposure in the Antarctic limpet

Introduction

The Antarctic limpet Nacella concinna, key species on the shores of the Western Antarctic Peninsula (AP) and nearby South Shetland and South Georgia archipelagos, colonizes intertidal and shallow sub-littoral habitats down to 130 m. A subgroup of the limpet population with distinguishable shell morphology [START_REF] Nolan | Size, shape and shell morphology in the Antarctic limpet Nacella concinna at Signy Island, South Orkney Islands[END_REF][START_REF] Walker | Introduction to the ecology of the Antarctic limpet Patinigera polaris at Signy Island, South Orkney Islands[END_REF] migrates upwards during the Austral spring, presumably to exploit the newly sprouting algal propagules in the intertidal [START_REF] Zacher | Grazer and UV radiation effects on an Antarctic intertidal microalgal assemblage: a long-term field study[END_REF]. This "intertidal subpopulation" tolerates fluctuations of environmental factors such as temperature and salinity on the high rocky shores, and limpets are therefore regarded as comparatively stress resistant among Antarctic stenotherms [START_REF] Peck | Prospects for surviving climate change in Antarctic aquatic species[END_REF]. In the context of the current massive climatic change happening in the AP region [START_REF] Vaughan | Recent trends in melting conditions on the Antarctic Peninsula and their implications for ice-sheet mass balance and sea level[END_REF], species like Nacella concinna are studied as models for the response of Antarctic stenotherms to environmental change, a response which encompasses behavioural, ecological and physiological traits within a single species. We want to clarify, how important the split is into two morphologically distinguishable subpopulations, one remaining sub-littoral and the other one "going intertidal". In addition to the conspicuous differences in shell morphology between the two subpopulations, behavioural traits differ such as the capacity to regulate the PO 2 in shell water [START_REF] Weihe | Differences in the physiological response of inter-and subtidal Antarctic limpets (Nacella concinna) to aerial exposure[END_REF].

Many Antarctic shelf species are extremely eurybathic with wide distribution on different shore levels or seasonal migrations between depth horizons [START_REF] Brey | Do Antarctic benthic invertebrates show an extended level of eurybathy?[END_REF]. Thus, intertidal N. concinna were reported to retreat to sub-littoral areas for the winter to mingle with the sub-littoral subpopulation before reproduction and recruitment take place in early spring [START_REF] Stanwell-Smith | The timing of reproduction in the Antarctic limpet Nacella concinna (Strebel, 1908) (Patellidae) at Signy Island, in relation to environmental variables[END_REF]. Indeed, new observations of scientists overwintering at the British Rothera station on Adelaide Island, West Antarctic Peninsula, report that a few Nacella concinna individuals survive the winter in intertidal rock crevices [START_REF] Waller | Ecophysiological strategies of Antarctic intertidal invertebrates faced with freezing stress[END_REF], and at King-George Island (KGI) some very small individuals of <12 mm shell length are found in the intertidal during early November (D. Abele and G. Husmann, pers. observation). These observations raise the question whether seasonal migration is indeed a species phenomenon that works similarly at all N. concinna residence sites along the AP. It is possible that there may be two differently adapted limpet populations drifting further apart under the impact of the ongoing climatic change. Very subtle evidence has been put forward documenting the existence of two genetically separating groups locally at King-George Island. These investigations of ISSR markers (inter simple sequence repeats) saw differences only during spring and early summer when migration is at a seasonal standstill and intertidal and sub-littoral limpets are most clearly separated [START_REF] Aranzamendi | Genetic differentiation between morphotypes in the Antarctic limpet Nacella concinna as revealed by inter-simple sequence repeat markers[END_REF].

Both subpopulations would be differing not only with respect to their physiological statusquo, especially when sampled in spring and summer on different shore levels, but moreover would presumably differ with respect to their general stress response capacities, resulting from their different environmental adaptation. Limpets colonizing the high intertidal and surf zone are daily exposed to air or caught in confined water bodies of the tide pools in one of the most demanding rocky shore environments in the world. Temperature can fluctuate up to 10°C during daytime low tides, whereas during the night new ice may form on the rocks.

Snow and ice melt waters were shown to be extremely stressful for the limpets, which clamp their shells down to the rock in order to avoid fresh water exposure [START_REF] Davenport | Meltwater effects on intertidal Antarctic limpets[END_REF].

Compared to this, the sub-littoral habitats, even under the influence of climate change are comparably stable with temperature fluctuating between -1.8 and + 1.5C and salinity between 34.1-34.2 ‰ over the year (O. Gonzalez, Argentine Antarctic Institute, unpubl. data of temperature andsalinity in Potter Cove, KGI between Sept 2007 andDecember 2008 in 20 m water depth). However, other factors such as increased input of lithogenic particles from underneath melting glaciers and from eroding land surfaces during the austral summer [START_REF] Ahn | Diatom composition and biomass variability in nearshore waters of Maxwell Bay, Antarctica, during the 1992/1993 austral summer[END_REF][START_REF] Klöser | Hydrography of Potter Cove, a small fjord-like inlet of King George Island (South Shetlands)[END_REF][START_REF] Yoo | Water column properties and dispersal pattern of suspended particulate matter (SPM) of Marian Cove during austral summer, King George Island, West Antarctica[END_REF] are bound to primarily affect the sub-littoral limpet fauna in the coastal environment of this volcanic archipelago [START_REF] Ahn | A baseline study on metal concentrations in the Antarctic limpet Nacella concinna (Gastropoda: Patellidae) on King George Island: variations with sex and body parts[END_REF].

Plumes of volcanic rock sediments carry high concentrations of heavy metals including Fe, Al, Cu and Zn into the nearshore waters of the South Shetland Archipelago [START_REF] Abele | Iron, copper and manganese discharge from glacial melting into Potter Cove and metal concentrations in Laternula elliptica shells[END_REF][START_REF] Dick | Is the umbo matrix of bivalve shells (Laternula elliptica) a climate archive[END_REF], and similar phenomena are described from areas along the maritime AP [START_REF] Dierssen | Glacial meltwater dynamics in coastal waters west of Antarctic Peninsula[END_REF]. Once taken up by sedimentary grazers or benthic filter feeders, these metals can exacerbate oxidative stress in animal tissues. Fe 2+ is a well known Fenton reactant which, if not tightly bound to ferritin Fe-storage protein, catalyzes the reduction of H 2 O 2 to the detrimental hydroxyl radical (OH•) and hydroxyl anion (OH) [START_REF] Gonzalez | Iron and radical content in Mya arenaria, possible sources of NO generation[END_REF]. The resulting Fe 3+ can be reoxidized mainly by interaction with O 2

•-released in small amounts by the mitochondria, or in large quantities during oxidative burst reactions.

Aluminium (Al 3+ ) is the most abundant metal in the earth crust and although not a transition metal and Fenton reactant, exacerbates Fe 2+ catalyzed lipid peroxidation in animal tissues [START_REF] Quinlan | Action of lead(II) and aluminium (III) ions on iron-stimulated lipid peroxidation in liposomes, erythrocytes and rat liver microsomal fractions[END_REF]. Likewise, Zn is not in itself a ROS producing metal, but when highly concentrated it can damage the mitochondria and induce higher rates of O 2 •-release [START_REF] Halliwell | Free Radicals in Biology and Medicine[END_REF]. The pro-oxidant effect of the non-essential element Cd is mainly via depletion of glutathione and protein-bound sulfhydryl groups, resulting in enhanced production of reactive oxygen species (ROS) [START_REF] Stohs | Oxidative mechanisms in the toxicity of metal ions[END_REF].

To test for differences in physiological regulatory capabilities, we have conducted air and (aquatic) hypoxia exposure experiments with both limpet subpopulations at King-George Island (KGI, South Shetland Islands). As a first set of results we described how intertidal limpets minimize water loss, how they manage to remain active, maintain energetic homeostasis and avoid tissue hypoxia and accumulation of anaerobic metabolites during exposure to dry air, in comparison to their sub-littoral conspecifics [START_REF] Weihe | Differences in the physiological response of inter-and subtidal Antarctic limpets (Nacella concinna) to aerial exposure[END_REF].

Material and Methods

Sample collection

Intertidal Nacella concinna were collected during low tide at with respect to water loss and physiological parameters (see also [START_REF] Weihe | Differences in the physiological response of inter-and subtidal Antarctic limpets (Nacella concinna) to aerial exposure[END_REF].

Contrary to other works [START_REF] Kensler | Desiccation resistance of intertidal crevice species as a factor in their zonation[END_REF], we did not attempt to measure natural survival times or LT50 values of air exposed limpets. Experiments were started in the evening, as the animals were observed to be more active during the day, when they left the dishes to crawl around in the desiccator. No animal died during air exposure.

For the hypoxia incubations animals were placed in 2L glass jars for 2, 6, 12, 24 and 48h.

Each time-group consisted of either 15 intertidal or 10 sub-littoral animals. The jars were cooled to 0°C in a thermostated water bath, and the water in the jars was bubbled with a gas mixture containing 2% oxygen (= 2 kPa). Oxygen concentration was controlled using a fiber optical respirometer (Precision Sensing GmbH, Regensburg, Germany).

Tissue samples for biochemical analyses were snap frozen and stored in liquid N 2 at Jubany-Dallmann. Samples were transported to Bremerhaven, Germany and stored in liquid N 2 until analysis.

Heavy metal measurement

The Fe and Cd, Zn and Al concentrations were measured in digestive gland and gill tissues of animals collected in 2005. The lyophilised samples were digested in 5 ml subboiled HNO 3 (65%) and 1 ml subboiled HF (40%) for 2 hours at 60°C, 3h at 100°C and 5 hours at 160° in closed teflon beakers. Afterwards the samples were heated in 5 ml ultrapure H 2 O for 5 hours at 160°C with the lid not firmly closed to evaporate acid and water. Subsequently, sample volume was adjusted to 10 ml with 1 molar subboiled HNO 3 . Fe analysis was carried out by inductively coupled plasma optical emission spectrometry (ICP/OES, IRIS Intrepid Typ Duo, Thermo Nicolet GmbH) at 259,9 nm, Al at 396,1 nm, and Zn at 206,2nm [START_REF] Nölte | ICP Emission Spectrometry: A Practical Guide[END_REF]. Cd was measured in the same samples by GF-AAS (graphite furnace atomic absorption spectrometry) (Zeeman 4100-Perkin Elmer) [START_REF] Schlemmer | Analytical Graphite Furnace Atomic Absorption Spectrometry: A Laboratory Guide[END_REF].

Calibrations were performed for ICP-OES measurements with multi-element solutions (Merck IV) in a range from 100 µg L -1 to 10 mg L -1 . Cd calibration was in a range from 0.1 µg L -1 to 2 µg L -1 .

Measurements of antioxidant enzyme

Superoxide dismutase (SOD) activity was measured in aliquots of 30 -180 mg of frozen gill and digestive gland tissue that were ground in liquid nitrogen and homogenised with a micropistill in Tris buffer (20 mM TRIS-HCl, 1mM EDTA, pH 7.6) at 1:4 (gill) and 1:6

(digestive gland) (w/v). Samples were centrifuged for 3 min at 18 000g and at 4°C. SOD activity was measured as degree of inhibition of the reduction of cytochrome c by superoxides generated by a xanthine oxidase/xanthine system at 550 nm in 43 mM potassium buffer with 0.1 mM EDTA, pH 7.8 according to [START_REF] Livingstone | Antioxidant enzymes in the digestive gland of the common mussel Mytilus edulis[END_REF]. 1 Unit SOD causes a 50% inhibition under the assay conditions. Mitochondrial and cytosolic SOD isoforms were not distinguished. Catalase (CAT) activities were determined in the same extracts. The activity was determined by recording the time of H 2 O 2 decomposition, resulting in a decrease of absorbance from 0.45 to 0.4 at 240 nm (1 unit) after [START_REF] Aebi | Catalase in vitro[END_REF] Determination of reduced (GSH) and oxidized (GSSG) glutathione by HPLC

The glutathione status represents the most important determinant for the cellular redox environment. The content of GSH and GSSG was determined according to [START_REF] Fariss | High-performance liquid chromatography of thiols and disulfides: dinitrophenol derivatives[END_REF]. Frozen tissue was ground in liquid nitrogen and homogenised in 1:10 (w/v) pre-cooled PCA (10% containing 2 mM bathophenanthroline-disulphonic acid) bubbled with nitrogen.

After centrifugation at 15 000 g for 5 min at 4°C, 500 µl of the supernatant were mixed with 10 µl pH-indicator (1 mM m-cresol purple sodium salt containing 0.5 M iodoacetic acid (IAA)). 50 µl 1 mM γ-glutamyl-glutamate (in 0.3% PCA) was added as internal standard. The pH was adjusted to 8.5 with 5 M KOH (containing 0.3 M N-morpholine-propanesulfonic acid). The mixture was incubated at room temperature for 45 min, to allow IAA to bind GSH.

Subsequently samples were centrifuged for 5 min at 15 000 g and 4°C. 300 µl of the supernatant were added to the double amount of 1% 1-fluor-2,4-dinitrobenzene (diluted in 100% ethanol, HPLC grade) and derivatized in dark vials at room temperature over 24 h.

Samples were stored in dark HPLC vials at -20°C. Prior to measurement, thawed samples were centrifuged at 7500 g for 1 min at 4°C and filtered through 0.2 µm nylon membrane filters. HPLC sample separation was carried out on a Beckmann Coulter HPLC System using a NH 2 -spherisorp column, 5 µm 240 x 4 mm (Waters, Germany). Solvent A: 80% methanol and solvent B: 20% sodium acetate stock and 80% solvent A. Sodium acetate stock was prepared by dissolving 272 g sodium acetate trihydrate in 122 ml Milli-Q water and 378 ml of concentrated HPLC-grade acetic acid. The gradient program was as follows: 3 min hold at 92% A followed by a 28 min linear gradient to 40% A and 15 min re-equilibration phase.

Flow rate was 1.2 ml min -1 at 2.3 to 2.8 psi backpressure. Peaks were recorded with a photodiode array detector at 365 nm.

Tissue redox potential (E mV ) can be calculated based on Nernst's equation as a function of the total glutathione concentration [GSH], the glutathione redox ratio (GSSG/GSH), and the tissue specific pH [START_REF] Schafer | Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple[END_REF].

Tissue pH

We measured the pH in the gills of control and air exposure groups and in the foot tissue of control animals. Nacella concinna foot tissue is buffered by CaCO 2 and, therefore, pHchanges cannot be reliably measured in this tissue [START_REF] Pörtner | Intracellular pH and energy metabolism in the highly stenothermal Antarctic bivalve Limopsis marionensis as a function of ambient temperature[END_REF] for comparison between groups.

Tissue pH was determined using the homogenate technique in a system thermostated at 4°C, the lowest operable temperature. Prior to measurements the pH electrode (SenTix Mic, WTW, Germany) was calibrated at 4°C with precise calibration solutions (AppliChem Darmstadt; pH 6.865-A1259; pH 7.413-A1260). Readings of pH were recorded on a Kipp & Zonen chart recorder. For tissue measurements, foot and gill tissue (100-200 mg) was ground in liquid nitrogen and the powder added to a 0.5 ml eppendorf cup containing 0.15 ml of medium composed of 160 mM potassium fluoride, 2 mM nitrilotriacetic acid. The cup was closed after layering with air bubble free medium, and the tissue homogenised by ultrasound (Brandson sonifier 450, duty cycle 40%, output control 8) at 0°C and centrifuged at 20 000 g at 4°C for 30 sec. For most samples, the pH values were used to calculate the tissue redox potential. In cases, where sample size was too small, to measure the glutathione concentration and tissue pH, a mean pH of the corresponding experimental group was used to calculate the tissue redox potential.

Ascorbate and malondialdehyde

Ascorbate and malondialdehyde (MDA) concentrations were measured after [START_REF] Lazzarino | Single-sample preparation for simultaneous cellular redox and energy state determination[END_REF] using HPLC. Samples were prepared as described in [START_REF] Weihe | Differences in the physiological response of inter-and subtidal Antarctic limpets (Nacella concinna) to aerial exposure[END_REF] and separated using a Kromasil column (250x4.6mm, 5 µm, Eka Chemicals, AB, Bohus, Sweden)

and its own guard column. Injection volume was 50 µl of undiluted extract. HPLC conditions (solvents, gradient, flow rate, detection) were applied as described in [START_REF] Lazzarino | Single-sample preparation for simultaneous cellular redox and energy state determination[END_REF].

Calibration was carried out using an ascorbate (Applichem A1052) and a MDA (Merck 805797) standard. Ascorbate was diluted in water and MDA dissolved in 1% sulphuric acid and then diluted with water. Calculations of sample concentrations were done using 32 Karat Software 7.0 (Beckmann Coulter, Krefeld, Germany).

Statistical analysis

Statistical analysis was performed using GraphPad Prism 4 Software.

Results

Differences in heavy metal concentrations and tissue antioxidant levels between intertidal and sub-littoral limpet populations

Heavy metal concentrations in gill (a) and digestive gland (DG, b) of sub-littoral and intertidal limpets are shown in Table 1. Metal concentrations in gills did not differ statistically between subpopulations except iron (p = 0.0076). However, all metals were more concentrated in sub-littoral than intertidal DG and, with the exception of Cd, this difference was highly significant. Especially iron and aluminum were much more concentrated in sublittoral than intertidal limpet digestive glands.

Under control conditions (submerged) both antioxidant enzymes were more active in gills of intertidal specimens. Especially the SOD activity was twice as high compared to gills of sublittoral limpets (Table 2). By contrast, SOD activity in DG of intertidal limpets was 40% lower than in sub-littoral specimen DG, whereas DG catalase activity was the same in both limpet subpopulations (Table 2). Glutathione concentration, the ratio between oxidized (GSSG) and reduced (GSH) glutathione, and tissue ascorbate (vitamin C) concentrations were the same range in intertidal and sub-littoral limpet gills and also in foot tissue (Table 3).

Glutathione levels in DG were not analyzed.

Effects of hypoxic exposure on tissue antioxidant parameters

Hypoxic exposure of limpets over 2h and longer caused a significant decrease in intertidal limpet gill SOD activities (Fig. 1a). Sub-littoral animals had low gill SOD activity in the 0h hypoxia group (= submerged and normoxic), which increased significantly until 12h of hypoxia, but returned to control levels after 24h and 48h of hypoxia. Gill catalase activity remained stable over the entire time of hypoxia exposure in both groups (Fig. 1b). Values were always slightly higher in intertidal animal gills, a difference which became significant only in the 24h group. Gill glutathione concentrations (GSH+2GSSG) were not influenced by hypoxic exposure in either of the two subpopulations, and furthermore no difference was observed between intertidal and sub-littoral gill glutathione content during hypoxia (data not shown). Gill pH values (data not shown) were very variable in both subpopulations throughout the hypoxic exposure. In neither subpopulation did gill tissues become clearly acidified, as we had, indeed, expected. Tissue pH and glutathione values were used to calculate changes of gill redox potential according to Nernst's equation and results are shown in Fig. 1c. Gill redox potential was significantly more oxidized (-333 ± 6.97 mV) in the intertidal than the sub-littoral (-359 ± 4.03 mV) control group (0h) and was rapidly reduced during hypoxia with significant effects after 2h and 12h at 2% oxygen. Gill tissues of sublittoral limpets were generally more reduced throughout the hypoxic exposure without significant change over time.

Hypoxic exposure caused significant decrease in digestive gland SOD activities of both subpopulations to significantly lower levels after 2h (sub-littoral) and 6h (intertidal) (one-way ANOVA, intertidal p = 0.0002; F = 6.076, n = 37; sub-littoral: p < 0.0001, F = 13,87, n = 36, Fig. 2a). Hypoxia effected CAT activity only in intertidal limpet DG (p = 0.0011; F = 5363; one-way ANOVA) (Fig. 2b). CAT activity in sub-littoral DGs remained unchanged during hypoxic exposure, which led to a highly significant interaction term of hypoxia and subpopulation (p = 0.0005; F = 5.171).

Hypoxia exposure of limpets over 48 h caused no change in total glutathione (GSH+2GSSG, data not shown) or ascorbate concentrations in limpet foot muscle. Ascorbate values in intertidal and sub-littoral limpets (only intertidal shown in Fig. 3) were very variable and, if anything, declined over the 2 days at 2 kPa PO 2 , but the trend was not significant. The glutathione redox ratio GSSG/GSH was more variable and significantly influenced by hypoxia in sub-littoral (p = 0.0383; KW = 11.75; one-way ANOVA) than intertidal foot muscle (Fig. 4) with no significant change over time of hypoxia exposure. Both subpopulations differed significantly concerning their redox ratio (subpopulation p = 0.0263; F = 5.32; interaction p = 0.0055; F = 3.918; n = 52; two-way ANOVA). We did not measure antioxidant enzyme levels in limpet foot muscles. Concentrations of malondialdehyde (MDA)

were a little higher in intertidal (0.01 ± 0.07 µmol g -1 fwt) than sub-littoral animal foot muscles (0.007 ± 0.003 µmol g -1 fwt), but the difference was not statistically significant (Mann-Whitney test, p = 0.081, U = 142, n = 39).

Effects of air exposure on tissue antioxidant parameters

High SOD values in gills of the intertidal control animals were significantly reduced during 6h air exposure and equalled the values in sub-littoral 6h-group (p > 0.05, Fig. 5a). Between 6 and 12h of air exposure, gill SOD activity increased in both groups (significant only in sublittoral p < 0.05). In 24h air exposed limpets, gill SOD values were all back to low levels, resembling the controls in the sub-littoral group, significantly lower with respect to the high 12h-value, and also much lower than the control values of the intertidal limpets (p < 0.001, Tukey HSD). Thus, gill SOD activities decline in intertidal and, following a short phase of mild induction, also in sub-littoral limpets during air exposure. After 12h CAT activity became significantly lower in gills of sub-littoral limpets than intertidal specimens, which kept CAT activity constant (two-way ANOVA, subpopulation p < 0.0001, F = 23.87, n = 54, Fig. 5b).

Alterations within the concentration of glutathione (GSH+2GSSG, Fig. 6a) in limpet gills during air exposure were significantly different between subpopulations in the overall twoway ANOVA (p = 0.0078; F = 7.838; n = 46). Air exposure had a significant effect on total glutathione concentration (p = 0.0103; F = 4.289, two-way ANOVA). Pair wise comparisons did not yield significant differences between both subpopulations at any time period. Gill glutathione in intertidal animals remained stable and even increased slightly throughout the exposure. In contrast, sub-littoral animal gill glutathione declined from 511.9 ± 86.02 nmol g -1 fwt (n = 8) in controls to 335.2 ± 118.2 nmol g -1 fwt during the first six hours of air exposure, The ascorbate concentration in limpet foot muscle did not change during air exposure (twoway ANOVA p > 0.05) and did not differ between subpopulations (intertidal 0.104 ± 0.09 µmol ascorbate g -1 fwt; n = 27; sub-littoral 0.093 ± 0.06 µmol ascorbate g -1 fwt; n = 27; unpaired t-test p = 0.5949). Malondialdehyde (mean ± SD) was more concentrated in foot muscle of intertidal limpets (0.00869 ± 0.002 µmol g -1 fwt, n = 29) than of their sub-littoral congeners (0.00679 ± 0.003 µmol g -1 fwt, n = 29; p = 0.0066; F = 8.066, two-way ANOVA, n = 58 samples, DF = 1).

Discussion

Heavy metal concentrations and oxidative stress in N. concinna

Absorbed metals in N. concinna are at least transiently stored and concentrated in the digestive gland [START_REF] Ahn | A baseline study on metal concentrations in the Antarctic limpet Nacella concinna (Gastropoda: Patellidae) on King George Island: variations with sex and body parts[END_REF][START_REF] Ahn | Influence of glacial runoff on baseline metal accumulation in the Antarctic limpet Nacella concinna from King George Island[END_REF][START_REF] Moreno | Heavy metals in Antarctic organisms[END_REF] [START_REF] Ahn | Influence of glacial runoff on baseline metal accumulation in the Antarctic limpet Nacella concinna from King George Island[END_REF], resulting in similar concentrations in sub-littoral and intertidal limpets (gills and DG). Dissolved metals can be absorbed from the water column directly into molluscan gills (P. Gonzalez unpublished experimental data with dissolved Fe) and our present data document this form of uptake also for Fe and Al, the two most abundant metals in the KGI environment. However, primary enrichment of metals in sub-littoral limpets clearly occurs through deposit feeding. Migration of limpets to the intertidal at the beginning of the melting season in spring may thus help to reduce the risks related to heavy metal enrichment. However, the different heavy metal loads in both subpopulations persisted also after more than 2 weeks of maintenance in the same holding system without sediment, and consequently de-enrichment will take much longer, if at all it happens during summer.

Response of the antioxidant system of intertidal and sub-littoral N. concinna to hypoxia and aerial exposure

Intertidal limpets are more mobile and active with respect to shell water ventilation when submerged [START_REF] Weihe | Differences in the physiological response of inter-and subtidal Antarctic limpets (Nacella concinna) to aerial exposure[END_REF]. Oxygen consumption is possibly higher and additional oxidative stress arises from the changing oxygen availability during tidal cycles. Especially during low tide aerial exposure, gills are a main target for oxidative injury [START_REF] Malanga | Oxidative stress in gills of limpets from the Beagle Channel[END_REF], and consequently gills of intertidal limpets must be better protected from oxidative stress by antioxidant enzymes. The oxidative stress during low tides arises either from breathing (gaping) air in intertidal Nacella concinna, or from hypoxia during shell contraction and subsequent reoxygenation. It is typical that the antioxidant response under such variable conditions is catalyzed through the enzymes SOD and CAT for a rapid, ad-hoc response to change, whereas the glutathione status, a basal buffer of tissue redox potential does not differ between intertidal and sub-littoral limpets.

Response to aerial exposure and hypoxia in the oxidative stress parameters differed in intertidal N. concinna, whereas the antioxidant enzyme activities in sub-littoral specimens responded in the same way to both forms of stress (Table 4). This fits with our observation that intertidal limpets respond more rigorously to air exposure than sub-littoral limpets, which maintained shell water PO 2 at low levels independently of submergence state. Whereas sublittoral limpets responded equally to air-exposure and hypoxia, by inducing anaerobic metabolism, intertidal limpets differentiate between air exposure, during which they remain aerobic and hypoxia during which anaerobic metabolites accumulate (Weihe and Abele, 2008, Weihe unpublished data). The most obvious difference with respect to the antioxidants occurs in the gills. Intertidal limpets reduce (SOD) activities, whereas in sub-littoral limpets gill SOD activity increases under both forms of stress.

Our data support a concept formulated earlier [START_REF] Weihe | Differences in the physiological response of inter-and subtidal Antarctic limpets (Nacella concinna) to aerial exposure[END_REF] that the first strategy of intertidal limpets is to down regulate metabolism and save energy, including the synthesis of antioxidants, during air exposure/hypoxia. This is in keeping with stable glutathione concentration and maintenance of tissue reducing capacity (low redox potential at -350 to -355 mV), as well as stable pH in the gills of the intertidal animals throughout the whole time of air exposure. Up-regulation of gill SOD activities in sub-littoral animals exposed to air and hypoxia would then indicate a response to stress by induction of enzymatic antioxidant protection to maintain tissue redox potential low (E mV subtidal gill in Tab. 4). Glutathione levels diminished (6h) and the gills became transiently oxidized (12h) and acidified during air exposure, indicating that the sub-littoral specimens had more problems to maintain redox and pH homeostasis when exposed to desiccation, a previously not experienced state for them.

Metabolic down regulation during air exposure in intertidal animals included down regulation of CAT activity in DG, whereas DG CAT activity during hypoxia was maintained high after a short drop in the beginning. The risk of oxidative stress upon re-oxygenation is presumably too high in this trace metal loaded tissue.

Foot muscle tissue appears well buffered by glutathione, with more oxidized GSSG/GSH ratio during the first 12h of exposure in air exposed intertidal compared to sub-littoral limpets.

Especially in combination with significantly higher MDA levels in foot muscle of intertidal limpets, this indicates higher ROS production and lipid peroxidation in foot muscle of intertidal limpets during air exposure and may be suggestive of oxygen uptake happening directly over the surface of the foot during air exposure. Indeed, in our first paper we showed that intertidal limpet foot muscle is maintained aerobic during air exposure, whereas sublittoral limpets switch to anaerobic metabolism [START_REF] Weihe | Differences in the physiological response of inter-and subtidal Antarctic limpets (Nacella concinna) to aerial exposure[END_REF], again documenting insufficient adaptation for life under intertidal exposure conditions.

The most important conclusion from our study is therefore that physiological adaptation for survival in the Antarctic intertidal shapes limpet behavior including their biochemical and metabolic response to stress exposure. One important strategy seems to be metabolic down regulation of unnecessary enzyme activity and presumably a general metabolic reduction during stress exposure in intertidal limpets (gill and digestive gland) for the purpose of energy saving. Aerial respiration during low tide may further prevent the limpet foot muscle from becoming anaerobic and accumulating major amounts of lactate and short chained organic acids. The differences in stress response are not immediately lost upon maintenance of both limpet sub-groups under standardized conditions, and thus are not purely adaptive, but indicate fundamental differences in the metabolic strategies between the two limpet populations, which may perhaps have a genetic background [START_REF] Aranzamendi | Genetic differentiation between morphotypes in the Antarctic limpet Nacella concinna as revealed by inter-simple sequence repeat markers[END_REF].

However, it clearly indicates that some Antarctic marine invertebrates have the capacity to adjust their response and survive in changing Antarctic coastal environments. Only shared genetic and physiological research on biogeographical gradients can show whether genetic limitations exist to physiological flexibility and adaptive capacities in these species.

Tab Tab. 4: Changes of oxidative stress parameters in intertidal and sub-littoral (sub-lit) Nacella concinna upon exposure to air and hypoxia (2kPa). Arrows indicate increase (↑) or decrease (↓) of enzyme activity. In case of pHa and redox potential (E mV) "↓" indicates acidification/reduction. No change observed is indicated as "↔" and an initial decrease with subsequent slight recovery of a parameter by "↓ ↑ " and " ↓ ↑" stands for early transient decrease followed by recovery. intertidal sub-littoral

air hypoxia Air hypoxia SOD, gill ↓ ↓ ↑ ↑ CAT, gill ↔ ↔ ↔ ↔ E mV , gill ↔ ↓ ↓ ↑ ↔ pH, gill ↔ ↔ ↓ ↑ ↔ SOD, DG ↔ ↓ ↓ ↓ CAT, DG ↓ ↓ ↑ ↔ ↔

Figure Captions

  but the difference remained insignificant because of the low number of samples (6h-value, n = 3). Subsequently glutathione concentration increased also in the sub-littoral limpet gills (significant between 6h and 24h) but sub-littoral values remained below the values in the intertidal specimens.Tissue pH was measured in gills and foot tissue of sub-littoral and intertidal control animals in 2005 and 2007. After confirming that pH did not differ between control animals in both years, the values were pooled for each tissue and analysed for the general differences between subpopulations. In submersed control groups, gill tissue of intertidal Nacella concinna had a significantly lower pH (7.391 ± 0.064, n = 10) than sub-littoral limpet gills (7.707 ± 0.188, n = 8, T-test p = 0.0019). Changes of gill pH over time differed significantly between intertidal and sub-littoral limpets when exposed to air (interaction p = 0.001; F = 7.236; aerial exposure (time) p < 0.0001; F = 10.53; n = 16-19). This was mainly due to a dramatic decrease from pH 7.7 to 7.1 in sub-littoral limpet gills between controls and the 12h air exposure group (p < 0.05; ANOVA; Dunn´s Multiple Comparison Test, data not shown). Fig 6b depicts the resulting redox potential calculated according to Nernst's equation. The low number of samples in some groups is due to limitation of gill tissue for both analyses. Low pH and higher glutathione redox ratio (GSSG/GSH) in gills of intertidal specimens resulted in a redox potential of -350 mV in control animals, which was maintained throughout the time of air exposure (ANOVA, p = 0.8668; F = 0.241). Sub-littoral limpet gills were initially (0h) more reduced than intertidal gills at 0h and became transiently oxidized after 12h (one-way ANOVA, p < 0.05), before going back to reduced control values at 24 h of air exposure. High SOD activities in digestive gland of sub-littoral control limpets dropped significantly within 6h of experimental air exposure (Fig. 7b) and continued on low levels. No significant change of SOD activity occurred in DG of either subpopulation between 6 and 24h of air exposure. DG CAT activity decreased significantly in the intertidal subpopulation during air exposure and remained constant in sub-littoral animals (subpopulation p < 0.0001, F = 19.91, n = 49; interaction p = 0.024; F = 3.491; two-way ANOVA). The effect of air exposure time and tidal level (= subpopulation) on glutathione concentrations in the foot were significant (time: p = 0.0006; F = 7.155; tidal level: p = 0.0021; F = 10.92; n = 46) with an insignificant interaction between factors time and tidal level (interaction term: p = 0.2461; F = 1.441), indicating similar changes of glutathione concentration to occur in the foot muscle in both subpopulations during aerial exposure. After 24h, sub-littoral foot tissue had significantly more glutathione than the intertidal 24h-group (p < 0.001, ANOVA, Bonferroni PostHoc test). Fluctuation of the redox ratio (GSSG/GSH) differed significantly between intertidal and sub-littoral foot tissues during air exposure. After 12h the foot tissue of intertidal limpets had accumulated proportionally more GSSG per GSH and the redox balance was twice as oxidised (0.1252 ± 0.07) compared to sub-littoral limpet foot muscles (0.0602 ± 0.02) (p < 0.05; population p = 0.0446; F = 4.36; interaction p = 0.035; F = 3.225; n = 17 sublittoral, n = 18 intertidal, two-way ANOVA, Bonferroni).

Fig. 1a :

 1a Fig. 1a: SOD activity in gills of Nacella concinna (black intertidal; grey subtidal) during hypoxia. Means + SD, n = 5 -10. * significant difference from 0h-value; one-way ANOVA. Intertidal p < 0.0001; F = 12.55; n = 35; subtidal p = 0.0014; F = 5.102; n = 40. Subpopulations (p < 0.0001; F = 33.26) time of hypoxia incubation (p < 0.0001; F = 10.70) interaction (p < 0.0001; F = 10.97). # significant differences between subpopulations: significance level p < 0.001 between controls and p < 0.05 between 24-values.

Fig. 1b :

 1b Fig. 1b: CAT activity in gills of Nacella concinna (black intertidal; grey subtidal) during hypoxia. Means + SD, n = 5 -11. Subpopulation p = 0.001; F = 11.76, n = 81 two-way ANOVA. # significant difference between the subpopulations (two-way ANOVA, Bonferroni, p < 0.05).

Fig. 1c :

 1c Fig.1c: Gill tissue redox potential of Nacella concinna (black intertidal; grey subtidal) during hypoxia. Means + SD, n = 3 -7. * significantly more reduced than controls (p < 0.05 one-way ANOVA). # significant differences between subpopulations (p < 0.05 two-way ANOVA). Interaction: p = 0.0004; F = 5.576; population: p = 0.0125; 6.775. n = 57.

Fig 2a :Fig. 2b :

 2a2b Fig 2a: Superoxide dismutase activity in digestive glands of intertidal (black) and subtidal (grey) Nacella concinna during hypoxia incubation for several hours. Means + SD. N = 5 -9. * indicates significantly lower activities than in the controls; one-way ANOVA. ). Intertidal p = 0.0002; F = 6.976; n = 37; subtidal p < 0.0001; F = 13.87; n = 36 (one-way ANOVA). # significant difference between the subpopulations (two-way ANOVA, Bonferroni, p < 0.001). Time p < 0.0001; F = 20.25. subpopulations p = 0.0001; F = 16.58. Interaction p = 0.0031; F = 4.045; n = 73; two-way ANOVA Fig. 2b: Catalase activity in digestive glands of intertidal (black) and subtidal (grey) Nacella concinna during hypoxia incubation for several hours. Means + SD. N = 5 -10. Intertidal p = 0.0011; F = 5.363 (one-way ANOVA). Hypoxia p = 0.0376; F = 2.529; interaction p = 0.0005; F = 5.171 twoway ANOVA. # indicates significantly different values p < 0.01 Bonferroni.

Fig. 3 :Fig. 4 :Fig. 5a :

 345a Fig. 3: Ascorbate concentration in foot muscle of intertidal N. concinna exposed to 2%-oxygen.Fig. 4: Ratio of oxidised (GSSG) and reduced (GSH ) glutathione (GSSG / GSH) in foot tissue of the Antarctic limpet Nacella concinna (black intertidal; grey subtidal) during hypoxia. Subtidal p = 0.0383; KW = 11.75; n = 25 (one-way ANOVA). Means + SD. N = 3 -5. time p = 0.0414; F = 2.573; subpopulation p = 0.0263; F = 5.32 # indicates significant differences p < 0.01 (Bonferroni) ; interaction p = 0.0055; F = 3.918. n = 52; two-way ANOVA

Fig. 5b :

 5b Fig. 5b: Catalase activity in gill of the Antarctic limpet Nacella concinna from the intertidal (black) and the subtidal (grey) during several hours of air exposure. Mean + SD. N = 3 -10. # significant difference between the subpopulations (two-way ANOVA, Bonferroni, p < 0.05). Subpopulation p < 0.0001; F = 23.87; n = 54 (two-way ANOVA).

Fig. 6a :Fig. 6b :

 6a6b Fig. 6a: Glutathione concentration (GSH + 2GSSG) in gills of intertidal (black) and (grey) sub-littoral Nacella concinna during air exposure. Subtidal p = 0.0099; KW = 11.36. Subpopulation p = 0.0078; F = 7.838; time p = 0.0103; F = 4.289; n = 46. Means + SD. N = 3 -8.

Fig. 7a :

 7a Fig. 7a: Superoxide dismutase activity in digestive gland of intertidal (black) and subtidal (grey) Antarctic limpets Nacella concinna during several hours of air exposure. Mean values + SD. N = 6 -10. * significantly different SOD activity to control animals. (ANOVA, Tukey if normally distributed and Dunn´s Multiple Comparison Test if not, p < 0.05) subtidal p < 0.0001; F = 11.51; n = 24. # significant difference between the subpopulations (two-way ANOVA, Bonferroni, p < 0.001) interaction p = 0.0036; F = 5.235; time p < 0.0001; F = 13.58; n = 51 (two-way ANOVA).

Fig. 7b :

 7b Fig. 7b: Catalase activity in digestive gland of the Antarctic limpet Nacella concinna from the intertidal (black) and the subtidal (grey) during several hours of air exposure. Mean + SD. N = 3 -10. * significantly lower CAT activity than in control animals (ANOVA, Dunn´s Multiple Comparison Test, p< 0.05) intertidal p = 0.034; F = 13.65, n = 23. # significant difference between the subpopulations. Subpopulation p < 0.0001; F = 19.91; interaction p = 0.024; F = 3.491, time p = 0.0295; F = 3.306; n = 49 (two-way ANOVA, Bonferroni, p < 0.05).

Fig. 8a :

 8a Fig. 8a: Total glutathione concentration in foot muscle of intertidal (black) and (grey) subtidal Nacella concinna during air exposure. * significantly lower total glutathione concentration than in control animals (ANOVA, Dunn´s Multiple Comparison Test, p < 0.05). intertidal p = 0.0165; KW = 10.26; subtidal p = 0.0267; KW = 9.202 (one-way ANOVA). # significant difference between the subpopulations (two-way ANOVA, Bonferroni, p < 0.05). Subpopulation p = 0.0021; F = 10.92; time p = 0.0006; F = 7.155; n = 45. Means + SD. N = 3 -8.

Fig. 8b :

 8b Fig. 8b: GSSG/GSH ratio in foot muscle of intertidal (black) and (grey) subtidal Nacella concinna during air exposure. Means + SD. N = 3 -8. # indicates significant differences p < 0.05 (Bonferroni). Subpopulation p = 0.0446; F = 3.225; interaction p = 0.035; F = 4.36; n = 41 (two-way ANOVA).

Fig. 1b :Fig. 1c :Fig 2a :Fig. 2b :Fig. 3 :Fig. 4 :

 1b1c2a2b34 Fig. 1b: CAT activity in gills of Nacella concinna (black intertidal; grey subtidal) during hypoxia.Means + SD, n = 5 -11. Subpopulation p = 0.001; F = 11.76, n = 81 two-way ANOVA. # significant difference between the subpopulations (two-way ANOVA, Bonferroni, p < 0.05).

Fig. 5b :

 5b Fig. 5b: Catalase activity in gills of the Antarctic limpet Nacella concinna from the intertidal (black) and the subtidal (grey) during several hours of air exposure. Mean + SD. N = 3 -10. # significant difference between the subpopulations (two-way ANOVA, Bonferroni, p < 0.05). Subpopulation p < 0.0001; F = 23.87; n = 54 (two-way ANOVA).

  Fig. 6a: Glutathione concentration (GSH + 2GSSG) in gills of intertidal (black) and (grey) sub-littoral Nacella concinna during air exposure. Subtidal p = 0.0099; KW = 11.36. Subpopulation p = 0.0078; F = 7.838; time p = 0.0103; F = 4.289; n = 46. Means + SD. N = 3 -8.

Fig. 6b :

 6b Fig. 6b: Redox potential in gills of intertidal (black) and (grey) subtidal Nacella concinna during air exposure. * significantly lower than in control group (p < 0.05, ANOVA, Dunn´s Multiple Comparison Test). # significant difference between subpopulations (two-way ANOVA, Bonferroni, p < 0.05) interaction p = 0.001; F = 6.524; time p = 0.0013; F = 6.275; n = 50. Means + SD. N = 3 -8.

  Fig. 7b: Catalase activity in digestive gland of the Antarctic limpet Nacella concinna from the intertidal (black) and the subtidal (grey) during several hours of air exposure. Mean + SD. N = 3 -10. * significantly lower CAT activity than in control animals (ANOVA, Dunn´s Multiple Comparison Test, p< 0.05) intertidal p = 0.034; F = 13.65, n = 23. # significant difference between the subpopulations. Subpopulation p < 0.0001; F = 19.91; interaction p = 0.024; F = 3.491, time p = 0.0295; F = 3.306; n = 49 (two-way ANOVA, Bonferroni, p < 0.05).

Fig. 8b :

 8b Fig. 8a: Total glutathione concentration in foot muscle of intertidal (black) and (grey) subtidal Nacella concinna during air exposure. * significantly lower total glutathione concentration than in control animals (ANOVA, Dunn´s Multiple Comparison Test, p < 0.05). intertidal p = 0.0165; KW = 10.26; subtidal p = 0.0267; KW = 9.202 (one-way ANOVA). # significant difference between the subpopulations (two-way ANOVA, Bonferroni, p < 0.05). Subpopulation p = 0.0021; F = 10.92; time p = 0.0006; F = 7.155; n = 45. Means + SD. N = 3 -8.

  The present paper deals with the possibility of oxidative stress occurring during aerial exposure in both limpet subpopulations at KGI, which could arise when limpets start air gaping, or during hypoxia re-oxygenation. Basically, one would expect antioxidant enzyme activity of superoxide dismutase (SOD) and catalase (CAT) to become reduced during hypoxia or to possibly increase as animals gape air. We also analyzed the reduction status of the redox buffer glutathione and the concentrations of the antioxidant ascorbate over time in two different tissues of submerged controls and air exposed intertidal and sub-littoral specimens. Moreover, experimental hypoxia exposure (2 kPa PO 2 ) was conducted to

demonstrate whether the limpets merely close their shells and become hypoxic, or whether the intertidal subpopulation is capable of aerial respiration. Because oxidative stress is exacerbated by heavy metals possibly ingested during grazing of the sediment surface, we compared Fe, Cd, Zn and Al concentrations in digestive gland and gills of sub-littoral and intertidal specimens.

  in samples collected in 2007. Samples from 2005 were used for the antioxidant enzyme measurements and glutathione concentrations in air exposed limpets.

	Experimental set-up and tissue sampling
	To test the response to dry air exposure, animals were individually placed on small plastic
	dishes over silica drying pearls in a desiccator for 2h (only in 2007), 6, 12, and 24h. Each
	time-group consisted of either 10 intertidal or 8 sub-littoral animals. The desiccator was
	placed in snow and covered with dark cloth, to reduce light and wind impact and keep
	temperatures close to 0°C. With this experimental routine we obtained reproducible results

Peñon 1, Potter Cove, King George Island on two occasions in November 2005 and November 2007. SCUBA divers collected sub-littoral animals in 13 -15 m depth in Potter Cove at about 1 km from the intertidal sampling location. All animals were immediately transferred to the aquarium of Dallmann laboratory, Jubany Station, and held in aerated, 0°C cold seawater from the cove

for between 10 days and 3 weeks before air exposure experiments were started. In the aquaria both groups of animals were maintained permanently submersed in flow through aquaria, so that any differences in control parameters were not caused by acute exposure to different insitu conditions. Ascorbate and MDA concentrations and glutathione concentration under hypoxia were measured

  . 1: Metal content [µg * g-1 dwt] in gill and digestive gland tissue of the Antarctic limpet Nacella concinna. Data as means ± SD, n = 4 -6. Unpaired t-test (Mann-Whitney if not normally distributed). Glutathione [µmol * g -1 fwt] and ascorbate concentrations [µmol * g -1 fwt] in intertidal and sub-littoral Nacella concinna control animals from 2005.

	Tab. 3: intertidal	sub-littoral		p-value
	A) gill
	GSH	Intertidal 345.5 ± 61.04	Sub-littoral 390.2 ± 56.29	p-value	> 0.05
	A) gill GSSG	61.57 ± 51.36	64.42 ± 45.47		> 0.05
	Fe GSSG/GSH	335.2 ± 59.78 0.141 ± 0.06	731.7± 189.9 0.136 ± 0.07	0.0079	> 0.05
	Cd B) foot	6.2 ± 2.7	3.7 ± 0.6	0.2000
	Al GSH	96.54 ± 37.65 450.8 ± 61.58	79.46 ± 65.30 480.8 ± 144.4	0.5476	> 0.05
	Zn GSSG	43.04 ± 14.69 34.7 ± 16.9	44.70 ± 5.3 47.89 ± 26.22	0.6905	> 0.05
	B) digestive gland GSSG/GSH	0.068 ± 0.023	0.078 ± 0.023		> 0.05
	Fe Ascorbate	271.5± 65.99 0.10 ± 0.06	2669 ± 1595 0.08 ± 0.01	0.0159	> 0.05
	Cd	25.3 ± 7.0	30.7 ± 17.0	1.0000
	Al	162.2 ± 85.53	2365.14 ± 2188	0.0159
	Zn	74.95 ± 7.76	119.9± 14.25	0.0286
	Tab. 2: Superoxide dismutase (SOD) and Catalase (CAT) activities [U * mg -1 fwt] in gill and
	digestive gland tissue of the Antarctic limpet Nacella concinna. Values were normally
	distributed and significant differences tested by unpaired t-test. N = 7 -10, means ± SD
		Intertidal	Sub-littoral		p-value
	SOD (gill)	1.018 ± 0.1	0.554 ± 0.1		0.0001
	CAT (gill)	0.526 ± 0.15	0.348 ± 0.16		0.0190
	SOD (DG)	2.228 ± 0.69	3.623 ± 1.21		0.0110
	CAT (DG)	9.541 ± 2.89	9.693 ± 1.95		> 0.05

Acknowledgement:

We thank the Alfred-Wegener-Institute (Germany) and the Argentine Antarctic Institute (Buenos Aires, Argentina) for supporting our work at Jubany Station and in Dallmann Laboratory, and the Deutsche Forschungsgemeinschaft, grant no DFG Ab124/7 for financing the PhD position of E.W. We are indebted to Stefanie Meyer and Ilse Stölting for excellent technical assistance and to 2 referees for their constructive review of the ms.


Giuseppa Buscaino
email: giuseppa.buscaino@cnr.it

Francesco Filiciotto

Gaspare Buffa

Antonio Bellante

Vincenzo Di Stefano

Anna Assenza

Francesco Fazio

Giovanni Caola

Salvatore Mazzola

Impact of an acoustic stimulus on the motility and blood parameters of European sea bass (Dicentrarchus labrax L.) and gilthead sea bream (Sparus aurata L.)

Keywords: Acoustic pollution, Acoustic stimulus, European sea bass, Gilthead sea bream, Motility, Blood glucose, Blood lactate, Haematocrit value

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Impact of an acoustic stimulus on the motility and blood parameters of European sea bass () and gilthead sea bream () 1 Introduction

The impact of human activity on marine habitats can produce adaptive alterations and other significant changes in animals [START_REF] Mcintyre | Human Impact on the Oceans: The 1990s and Beyond[END_REF][START_REF] Myrberg | The effects of man-made noise on the behaviour of marine animals[END_REF][START_REF] Popper | Anthropogenic sound: Effects on the behaviour and physiology of fishes[END_REF]. In recent years, many studies have been carried out with the aim of evaluating the effects of anthropogenic acoustic disturbance on marine organisms [START_REF] Santulli | Biochemical responses of European sea bass (Dicentrarchus labrax L.) to the stress induced by offshore experimental seismic prospecting in the mediterranean sea[END_REF][START_REF] Sarà | Effect of shipping traffic on behaviour of bluefin tuna Thunnus thynnus[END_REF][START_REF] Scholik | Effects of underwater noise on auditory sensitivity of a cyprinid fish[END_REF], thus increasing the awareness of the damage done to animals exposed to human related underwater sounds (National Research Council, 2000;2003;[START_REF] Ross | Ship Sources of Ambient Noise[END_REF]. These sounds are associated with shipping, seismic surveys, sonar, recreational boating and many other anthropogenic sources that are known to induce several types of responses in fishes [START_REF] Bart | Underwater ambient noise measurements in aquaculture systems: a survey[END_REF][START_REF] Engås | Effects of seismic shooting on local abundance and catch rates of cod (Gadus morhua) and haddock (Melanogrammus aeglefinus)[END_REF][START_REF] Myrberg | The effects of man-made noise on the behaviour of marine animals[END_REF][START_REF] Popper | Effects of exposure to seismic airgun use on hearing of 3 fish species[END_REF]Sandstrom et al., 2005;[START_REF] Schwarz | Responses of Pacific herring, Clupea harengus pallasi, to some underwater sounds[END_REF][START_REF] Smith | Noise-induced stress response and hearing loss in goldfish (Carassius auratus)[END_REF]. Pacific herring (Harengus pallasi) exhibited alarm responses in reaction to motorboat noise [START_REF] Schwarz | Responses of Pacific herring, Clupea harengus pallasi, to some underwater sounds[END_REF]. [START_REF] Engås | Effects of seismic shooting on local abundance and catch rates of cod (Gadus morhua) and haddock (Melanogrammus aeglefinus)[END_REF] found that seismic shooting has effects on the local abundance and distribution of Gadus morhua and haddock (Melanogrammus aeglefinus). Smith et al. (2004) examined the short-and long-term effects of increased ambient noise on the behaviour and hearing of goldfish (Carassius auratus). They noted that goldfish exhibited an initial startle response with a rapid burst of erratic swimming followed by general increased swimming activity with the onset of an experimental noise (bandwidth ranging from 0.1 kHz to 10 kHz at 160-170 dB re 1 µPa total sound pressure level). [START_REF] Kastelein | Startle response of captive North Sea fish species to underwater tones between 0.1 and 64 kHz[END_REF] observed a behaviour response of sea bass exposed to pure tone signals ranging between 0.1 and 0.7 kHz at 0-30 dB above the hearing thresholds.

Since acoustic signals in the 100-500 Hz band are detected by many species of fish [START_REF] Popper | Sound detection mechanism and capabilities of teleost fishes[END_REF] and there is an increase in this low-frequency ambient noise as a result of increased international shipping [START_REF] Ross | Ship Sources of Ambient Noise[END_REF], it is safe to assume that these noises are having an impact on the welfare of many fish species. While the effects of such anthropogenic sounds on marine mammals have been described [START_REF] Myrberg | The effects of man-made noise on the behaviour of marine animals[END_REF]National Research Council, 2000;2003;[START_REF] Ross | Ship Sources of Ambient Noise[END_REF][START_REF] Richardson | Marine Mammals and Noise[END_REF], the impact of underwater noise on marine fish is not understood sufficiently. Further information is needed to evaluate or predict any negative effects [START_REF] Popper | Anthropogenic sound: Effects on the behaviour and physiology of fishes[END_REF].

Previous studies have shown that acoustic stimulation can affect fish behaviour, but the physiological consequences have hardly been studied. Some studies have shown that acoustic stimulation can produce metabolic changes in fish. [START_REF] Smith | Noise-induced stress response and hearing loss in goldfish (Carassius auratus)[END_REF] pointed out a significant modification of plasma cortisol and glucose levels in goldfish after exposure to white noise. The present study aims to investigate the motility and haematological responses of European sea bass and gilthead sea bream exposed to an experimental acoustic stimulus in a low frequency range using analyses of movement, blood glucose, blood lactate and haematocrit values. The correlation between movement and blood parameters was also investigated.

Material and methods

Study animals

The experiment was carried out from July to September 2008 at the Istituto per l'Ambiente Marino Costiero of Consiglio Nazionale delle Ricerche (IAMC-CNR) -Laboratories of Capo Granitola (Trapani, Italy) using 14 sub-adult European sea bass (Dicentrarchus labrax) weighing 189.4 ± 80 g with body lengths of 26.2 ± 3.3 cm, and 14 sub adult gilthead sea bream (Sparus aurata) weighing 172.6 ± 23.7 g with body lengths of 22.9 ± 0.9 cm.

Three months before the beginning of the experiment, upon arrival at the laboratories from the marine fish farm of Trappeto (Palermo, Italy), fish were placed in circular tanks (diameter: 3 m, depth: 1 m, volume: 5000 L) at a low stock density (5 kg/m 3 ) with re-circulated and filtered seawater. Fish were exposed to the natural photoperiod and fed daily with commercial dry pellets.

Feeding was stopped at least 48 h before the experiment.

Acoustic stimulus

Most fish are able to detect sound and the range of hearing is from 100 to 500 Hz [START_REF] Popper | Sound detection mechanism and capabilities of teleost fishes[END_REF]. Keeping this in mind, as well as the fact that acoustic energy produced by vessel traffic is more intense at low frequencies [START_REF] Ross | Ship Sources of Ambient Noise[END_REF], it was decided that an acoustic stimulus with a frequency band of 0.1-1 kHz would be used. A 1-second linear sweep was used to cover the frequency band. The linear sweep was repeated for 10 minutes without pause.

Experimental procedure

During the experimental phase, sea bass and sea bream were randomly assigned to control and test groups. For each test, the fish was transported to an experimental sea cage (see Fig. 3) located in a circular natural harbour (with a diameter of about 200 m and a depth of 3 m) and left there to acclimate.

A research cabin was placed 8 m away from the sea cage. The cabin housed the sound generator, as well as video and sound recording equipment.

One hour later, the specimen was recorded (both audio and video) for 10 minutes with an acoustic acquisition system and two underwater video cameras (model RE-BCC6L, DSE, Italy) mounted outside the cage. One camera was mounted on the middle of the cage and the other camera was mounted on top of the cage (Fig. 3) so that the entire cage was visible. During video and sound recording, individuals in the test group were exposed to an acoustic stimulus while members of the control group had no stimulus applied. The fish were then captured with a net to take blood samples. After this, the fish were transferred into a small tank and then released after recovery. The glucose, lactate and haematocrit levels were measured in the fish blood samples, while fish motility was evaluated using the video recordings (see paragraph 2.5).

Blood parameters and motility data recorded in the control and test group of sea bass and sea bream were statistically analysed to assess possible differences between the groups. Moreover, a statistical correlation was applied to the total number of movements and blood parameters. The entire work presented here complied with current regulations regarding animal experimentation in Italy.

Blood sampling procedure and chemical analyses

A standardized handling procedure for each sea bass and sea bream was applied in order to standardize the potential stress produced during the blood sampling. After 10 minutes of audio and video recording, the fish was captured with a net from the experimental cage and immediately anesthetized with 2-phenoxyethanol (0.4 ml L -1 ) in a 10-litre tank. Fish reached stage V of anaesthesia [START_REF] Summerfelt | Anaesthesia, surgery and related techniques[END_REF]) within 1-2 minutes after which they were weighed and measured. Later, blood samples were collected from the caudal vein (using a 2.5 ml syringe with a 22 G×1 1/2″ needle) for the immediate assessment of glucose and lactate on whole blood with a portable blood glucose analyser (Accu-Chek Active, Roche Diagnostics GmbH, Mannheim, Germany) and blood lactate analyser (Accusport, Boehringer Mannheim, Germany). The time between capture and blood collection was less than 5 minutes. A Select-A-Fuge Model 24 blood micro haematocrit centrifuge (Bio-Dynamics, Inc., Indianapolis, United States) running at 3600 rpm for 5 minutes was used to assess the haematocrit value.

Motility sampling procedure

Two grids were affixed on the sides of the sea cage monitored by the cameras, each comprising nine regions, to analyse the movements of fish along the three axes of space (see Fig. 3). The images from the two cameras were synchronized and fish movements were analysed from the video recordings. The transit of the specimen from one region to another region of the grid was recorded.

A focal animal sampling technique modified from [START_REF] Altmann | Observational study of behaviour: sampling methods[END_REF] was used to analyze the images in slow motion. For each minute of sampling, we recorded a value (as sum of the movement across the x, y, and z axes) for a total of 10 values for each specimen.

Statistical analysis

An unpaired t-test was used to determine significant differences in blood glucose, blood lactate, and haematocrit levels as well as differences in movement (as the total movement during each minute of sampling for all specimens) between the control groups and the test groups. A linear regression model (y = a + bx) was applied to the total movement during the 10 minutes of sampling and the values for the blood parameters (glucose, lactate and haematocrit) of each fish in order to determine the degree of correlation in the control and test groups. A P < 0.05 was considered statistically significant.

Results

Motility

The results obtained during the motility sampling procedure showed that the amount of movement observed in the test groups of sea bream and sea bass were significantly higher (P < 0.0001 and P < 0.001, respectively) than those of the control groups. The total movement for the control and test groups of sea bream and sea bass are shown in Table 1 while the mean values of total movement per minute and the statistical significance are shown in Figure 4.

Blood parameters

In sea bream, glucose levels were significantly lower (P < 0.005) in the test group than in the control group, with a difference of 34.41 mg dL -1 . Blood lactate exhibited significantly increased values (P < 0.05) in the control group compared to the test group (difference of 1.13 mmol L -1 ).

In sea bass, noise exposure did not significantly affect glucose levels between the control and test groups (P < 0.94). Blood lactate showed significantly higher levels (P < 0.01) in the test group.

In both species, significantly higher levels of haematocrit were recorded for the test group compared with the control group (sea bream: P < 0.01; sea bass: P < 0.05), with an increase of 15.3% and 8.9% for sea bream and sea bass respectively. Mean blood glucose, blood lactate and haematocrit levels of sea bream and sea bass are shown in Table 1, while the mean values and the statistical significance are shown in Figure 5.

Correlation between movements and blood parameters

In the control groups of both species, the application of a linear regression showed no significant correlation between the values for total movement and the values for the blood parameters. The total value of the movement for the sea bream test group had a significant correlation with the values of blood glucose and lactate (Fig. 6, a andc). A significant correlation between the total movement and blood lactate levels was observed in sea bass (Fig. 6,d) while no significant correlation between movement and blood glucose was recorded in sea bream (Fig. 6,b).

Haematocrit levels and movement were highly correlated for both test groups (Fig. 6, e and f).

Discussion

The present study was conducted in a confined space, so that the obtained fish responses may be different to those observed in the wild. The analysis of sea bream and sea bass motility showed increased swimming activity, indicating a disturbance due to the acoustic stimulus. Although no changes were observed in the blood glucose levels of sea bass, the others blood parameters corroborated the motility observations for both species. The blood parameter changes and motility responses reflected an intense metabolic activity involving white muscle anaerobic fibres during the acoustic exposure.

The muscle metabolism of fish has been thoroughly investigated in the past [START_REF] Beamish | Swimming capacity[END_REF][START_REF] Brett | Physiological energetics[END_REF][START_REF] Fry | The effect of environmental factors on the physiology of fish[END_REF]. Fish, like other vertebrates, have several types of muscle fibre with different properties. Two fibre types are most important in locomotion: red or slow oxidative fibres (type IIb; low power system) and white or fast glycolytic fibres (type Ia; capacity-limited, highpower system).

Aerobic red muscle is used for routine swimming while anaerobic white muscle is used for bursts of activity (sprints and fast-start; [START_REF] Bennett | Anaerobic metabolism during activity in lizards[END_REF][START_REF] Goolish | Aerobic and anaerobic scaling in fish[END_REF][START_REF] Weiser | Anaerobic and aerobic energy production of young rainbow trout (Salmo gairdneri) during and after bursts of activity[END_REF].

Anaerobic pathways can also fuel intermediate or prolonged swims that can be sustained for few minutes, but these pathways eventually tire the fish [START_REF] Bone | Muscular and energetic aspects of fish swimming[END_REF][START_REF] Brett | Physiological energetics[END_REF]. In fact, white muscle glycogen reserves can be depleted by 50% in only 2 minutes of forced activity [START_REF] Goolish | Aerobic and anaerobic scaling in fish[END_REF]. The capacity for anaerobic energy production has been estimated from decreases in substrate reserves and end products accumulation with a conversion of glucose to lactate followed by stimulation of pyruvate metabolism and oxygen debt [START_REF] Goolish | Aerobic and anaerobic scaling in fish[END_REF][START_REF] Kauffman | Respiratory cost of swimming in larval and juvenile cyprinids[END_REF][START_REF] Puckett | Cost of sustained and burst swimming to juvenile coho salmon (Oncorhynchus kisutch)[END_REF]1985;[START_REF] Weiser | Anaerobic and aerobic energy production of young rainbow trout (Salmo gairdneri) during and after bursts of activity[END_REF]. Lactate produced in the myotome is released into the circulatory system following bursts of anaerobic activity.

In the present study, the statistically significant responses in motility and blood parameters (glucose and lactate) observed in both sea bass and sea bream exposed to the acoustic stimulus indicate increased muscle activity using anaerobic Ia fibres.

Furthermore, the increase of metabolic muscle activity implies a higher demand for oxygen, which is increased by increasing the respiratory rate. During this adaptive response, fish experience the haemopoietic activity of the spleen that encourages the production of red blood cells for oxygen transport [START_REF] Franklin | The role of the spleen during exercise in the antarctic teleost, pagothenia borchgrevinki[END_REF]. This condition leads to an increase in the levels of blood corpuscular components such haematocrit, one of the most reliable indexes. This increase was also found in the present study (Fig. 5).

The significant correlation between the movement values and the selected blood parameters (Fig. 6) supports what has been described above. However, the different blood glucose trends for the two species should be highlighted. Sea bream experienced a significant decrease in blood glucose levels and these levels were highly correlated with motility, while sea bass experienced no significant differences in glucose levels, and these levels were not correlated with motility. These responses may be explained by intrinsic species differences in recovery time and in the use of glucose for energy. These differences could also be due to a difference in sensitivity to sound exposure.

Conclusions

In conclusion, the results from this study showed that anthropogenic noise at low frequencies can influence the swimming activity of fish. Moreover, many previous studies have shown that muscle activity rates can be a very large part of the fish energy budget [START_REF] Boisclair | Testing assumptions of fish bioenergetics models by direct estimation of growth, consumption, and activity rates[END_REF][START_REF] Koch | Partitioning of energy in fish: can reduction of swimming activity compensate for the cost of production?[END_REF]. Consequently, increased swimming activity and the associated metabolic costs could compromise other biological activities, such as food acquisition, regulation due to environmental perturbation, migration and reproduction. [START_REF] Banner | Effects of noise on eggs and larvae of two estuarine fishes[END_REF][START_REF] Banner | Effects of noise on eggs and larvae of two estuarine fishes[END_REF][START_REF] Lagardère | Effects of noise on growth and reproduction of Crangon crangon in rearing tanks[END_REF] observed a drastic reduction of egg survival and reproductive and growth rates in farmed fish species exposed to high sound levels.

The present study shows the relationship between behaviour and haematological parameters in relation to noise exposure. It also shows that the use of different study approaches (physiological or behavioural) can lead to similar results. Moreover, our results indicate that our real-time field assays were appropriate for measuring the physiological impact of the acoustic stimulus in sea bass and sea bream. Although this short-term noise experiment showed an increase in fish motility, lactate and haematocrit values, it would be useful to carry out future tests with a similar experimental procedure on other fish species using longer-term noise exposure to better understand the impact of anthropogenic noise on fish behaviour and physiology.

  Figures 1 and 2.

6Figure 1 .

 1 Figure 1. Mean power spectrum of sweep signal and background noise. The sampling frequency of the signal was 100 kHz. The size of Fast Fourier Transform (FFT) was 1024 points and the window type was cosine tapered.

Figure 2 .

 2 Figure 2. Spectrogram of the sweep signal. Time in seconds and frequencies in hertz are shownalong the x-axis and y-axis, respectively. Amplitude was measured in dB (re 1µPa) using a colour scale. The sampling frequency of the signal was 100 kHz, the size of FFT was 2048 points and the window type was cosine tapered.

Figure 3 .

 3 Figure 3. A schematic view of the sea cage (1) with two underwater video cameras (2), a hydrophone inside the cage (3) and the underwater speaker (4).

Figure 4 .

 4 Figure 4. Mean values (± standard error of the mean, SEM) of total movement per minute for sea bream and sea bass with the statistical significance values.

Figure 5 .

 5 Figure 5. Mean values (± standard error of the mean, SEM) of blood glucose, lactate and haematocrit levels for sea bream and sea bass together with the statistical significance.

Figure 6 .

 6 Figure 6. Linear regression between the total number of movements and blood glucose (mg dL -1 ) (a), blood lactate ( mmol L -1 ) (c) and haematocrit levels (%) (e) in the gilthead sea bream test group; (b), (d) and (f) are the same measurements for the European sea bass test group. The dashed lines delimit the 95% confidence interval.

Table 1 .

 1 Total movement and means ± standard error of the mean (SEM) for blood glucose, blood lactate and haematocrit levels in the control and test groups of gilthead sea bream and European sea bass. SEM mean ± SEM mean ± SEM mean ± SEM Glucose (mg dl -1 ) 116.02 ± 7.42 81.61 ± 6.58 131.86 ± 5.47 132.61 ± 7.04 Lactate (mmol l -1 ) 1.92 ± 0.27 3.05 ± 0.38 13.54 ± 1.59 16.56 ± 1.80 Hematocrit (%) 18. 57 ± 2.30 33.88 ± 3.61 21.71 ± 2.2 30.57 ± 1.98

	Table 1
		Gilthead sea bream	European sea bass
		control group test group control group test group
	Motility
	Total movements	2451	3182	648	889
	Blood parameters	mean ±

Acknowledgments

This investigation was financially supported by the detached branch of the Capo Granitola of the IAMC-CNR, through funds made available from the "Acoustic Dispositive for the Reduction of Fishery and Marine Mammals Interaction -DAIMAR" project. The authors would like to thank Dr.

Paolo Domenici for his valuable comments on the manuscript and Dr. Antonino Adamo for his assistance with language revision.