A novel field transplantation technique reveals intra-specific metal-induced oxidative responses in strains of Ectocarpus siliculosus with different pollution histories

Environmental Pollution 199 (2015) 130e138

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A novel field transplantation technique reveals intra-specific metalinduced oxidative responses in strains of Ectocarpus siliculosus with different pollution histories
ez a, b, c, Alberto Gonza
lez d, Rodrigo A. Contreras d, A. John Moody e, Claudio A. Sa d Alejandra Moenne , Murray T. Brown a, * a

School of Marine Science & Engineering, Faculty of Science and Environment, Plymouth University, Drake Circus, PL4 8AA, Plymouth, United Kingdom Departamento de Medio Ambiente, Facultad de Ingeniería, Universidad de Playa Ancha, Casilla 34-V, Valparaíso, Chile ~ a #450, Vin ~ a del Mar, Chile Centro de Estudios Avanzados, Universidad de Playa Ancha, Traslavin d Departamento de Biología, Facultad de Química y Biología, Universidad de Santiago de Chile, Casilla 40 Correo 33, Santiago, Chile e School of Biological Sciences, Faculty of Science and Environment, Plymouth University, Drake Circus, PL4 8AA, Plymouth, United Kingdom b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 13 October 2014 Received in revised form 11 January 2015 Accepted 15 January 2015 Available online 30 January 2015

A novel field transplantation technique, in which seaweed material is incorporated into dialysis tubing, was used to investigate intra-specific responses to metals in the model brown alga Ectocarpus siliculosus. Metal accumulation in the two strains was similar, with higher concentrations in material deployed to the metal-contaminated site (Ventanas, Chile) than the pristine site (Quintay, Chile). However, the oxidative responses differed. At Ventanas, strain Es147 (from low-polluted site) underwent oxidative damage whereas Es524 (from highly polluted site) was not affected. Concentrations of reduced ascorbate (ASC) and reduced glutathione (GSH) were significantly higher in Es524. Activities of the antioxidant enzymes superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), and glutathione reductase (GR) all increased in Es524, whereas only SOD increased in Es147. For the first time, employing a field transplantation technique, we provide unambiguous evidence of inter-population variation of metal-tolerance in brown algae and establish that antioxidant defences are, in part, responsible. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Brown algae Metal accumulation Antioxidant metabolism Active biomonitoring

1. Introduction Beyond certain threshold concentrations, essential and nonessential metals can produce detrimental effects in marine algae, including photo-inhibition and disruption of electron transport chains in chloroplasts and mitochondria. As a consequence, there is over-production of reactive oxygen species (ROS) resulting in oxidative stress (Torres et al., 2008); for example, Cu excess disrupts electron transport chains in chloroplast and mitochondria generating over-production of the highly oxidizing
O 2 , the pre
lez et al., 2010). However, cursor of H2O2, a less reactive ROS (Gonza while exposure to excess metals can be harmful for many macroalgal species, some display inherent metal-tolerance (e.g. Brown et al., 2012; Ratkevicius et al., 2003), while others have evolved metal-tolerant ecotypes following long-term exposure to metal pollution (Contreras et al., 2005; Ritter et al., 2010). For example, * Corresponding author. E-mail address: [email protected] (M.T. Brown). http://dx.doi.org/10.1016/j.envpol.2015.01.026 0269-7491/© 2015 Elsevier Ltd. All rights reserved.

Nielsen et al. (2003) observed that growth rates of embryos and adults of Fucus serratus from a metal polluted location were significantly higher than those from a pristine location, when exposed up to 2 mmol L1 Cu in vitro. Although the mechanisms of tolerance have not been fully elucidated, there is evidence that the foundation for inter- and intra-specific variation in tolerance is a combination of efficient cell exclusion, synthesis of metal-chelating compounds and activation of reactive oxygen metabolism
lez et al., 2010; (Contreras et al., 2009; Gledhill et al., 1999; Gonza Mellado et al., 2012; Pawlik-Skowronska et al., 2007). Most of what is known about metal-mediated responses in seaweeds has been derived from experimentation under controlled conditions in the laboratory, but, despite these data being invaluable, they may not necessarily explain what is happening in the more complex natural environment. Therefore, if results from field investigations reflect those from laboratory studies greater credence can be accorded to the latter. In estuaries and coastal waters, seaweeds are the main primary producers at the base of trophic networks and provide habitat for a large diversity of other


ez et al. / Environmental Pollution 199 (2015) 130e138 C.A. Sa


ez et al., 2012b). Because of their ecological marine organisms (Sa importance and metal accumulation capacities brown seaweeds of the orders Fucales and Laminariales are the most widely used macroalgae for metal bioaccumulation studies (Brown and
ez et al., 2012a). Typically, assessment of metal Depledge, 1998; Sa pollution using seaweeds has involved a ‘passive’ bio-monitoring approach, whereby resident species are sampled and analysed for their metal content (e.g. Pawlik-Skowronska et al., 2007;
ez et al., 2012a). However, intrinsic and Ratkevicius et al., 2003; Sa extrinsic factors can influence the accumulation of some metals and population responses to metal exposure; the latter may result in differential tolerance and potentially create uncertainties in the interpretation of data leading to false environmental diagnoses (Brown et al., 2012). As an alternative procedure, several authors have suggested ‘active’ bio-monitoring as a better option, whereby individuals of species whose responses to metal-pollution are well researched are cultured in the laboratory and then transplanted to locations to be investigated (Brown et al., 2012; Chaphekar, 1991;
ez et al., 2012a). Advantages of this approach include inSa dividuals from the same population, or ramets of the same individual, can be transplanted into different locations, similar age individuals can be used, the time period of exposure is known and monitoring can be carried out even if the species is absent from a site. Of the active-bio-monitoring efforts so far reported most have
douin et al., 2008; Serisawa et al., involved fucoids and kelps (e.g. He 2002), but, their transplantation can be logistically complex and time-consuming due to their large stature, morphological complexity and relatively slow growth. Thus, the approach would benefit from improvements to the methodologies and using a wider range of species that should include morphologically less complex and faster growing species such as Ectocarpus siliculosus. E. siliculosus is a small filamentous brown alga that inhabits the low intertidal and shallow subtidal, growing free-floating or attached to hard substrate or epiphytic on larger seaweeds (Charrier et al., 2008). It has a global distribution in temperate coastal environments, and has been accepted as the model organism for the Phaeophyceae (Charrier et al., 2008). Our recent results

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from in vitro Cu exposure experiments, of up to 2.4 mmol L1 for 10 d, have established that strains with different pollution histories display divergent responses that involve cellular exclusion of Cu, production of metal chelators and antioxidant defences (Roncarati et al., 2015; S
aez et al., 2015). A field transplantation technique has been developed in which seaweed material is incorporated into dialysis tubing and then deployed to field stations with different levels of metal contamination, to investigate the responses of different strains of E. siliculosus in situ. Metal accumulation, oxidative damage and antioxidant responses were measured for this purpose. The aims of our investigation were threefold: a) to evaluate the effectiveness of the novel methodology and assess its suitability for incorporation into bio-monitoring programmes, b) identify mechanistic differences in stress responses between two strains of E. siliculosus with different pollution histories and c) to compare the results obtained in this field investigation with those from controlled laboratory experimentation.

2. Materials and methods 2.1. Transplantation experiment sites Two different locations in central Chile were chosen for the transplantation experiments, one polluted and one pristine. Due to the documented information of metal contamination in the loca
lez et al., 2008; Sa
ez et al., 2012a, 2012b), the Bay of tion (Gonza Ventanas (32 440 36.5500 S and 71290 35.7000 W) was chosen as the contaminated site (Fig. 1A); metal concentrations of up to 68 mg g1 have been recorded in subtidal sediments at this site
ez et al., 2012a). Sources of pollution come from Cu smelting and (Sa casting industries and thermoelectric complexes (Neaman et al., 2009), and from wastewaters of unknown origin delivered via an
ez et al., 2012a, 2012b). The pristine illegal sewage outfall (Sa location used was Quintay (3311046.1600 S and 7142018.7300 W) (Fig. 1A), a site with no history of metal pollution.

Fig. 1. (A) Map of the transplantation sites in central Chile, (B) diagram of the transplantation device, and (C) sample of the transplantation device in the field.

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ez et al. / Environmental Pollution 199 (2015) 130e138 C.A. Sa

To avoid proliferation of non-native E. siliculosus, the two strains used were native to Chile: Es524 (Culture Collection of Algae and Protozoa (CCAP) 1310/333), was isolated from Caleta Palito (Cha~ aral, Chile), a coastal site with a long history of high levels of Cu n pollution resulting from mining activities and Es147 (CCAP 1310/ 340), isolated from Caleta Coloso (Antofagasta, Chile), a harbour with a recent history of low metal inputs. The strains were cultured in 10 L polycarbonate bottles with clean autoclaved seawater and Provasoli nutrients (Provasoli and Carlucci, 1974), and cultured at 16  C, 14/10 of light/dark cycle, 45 mmol photons m2 sec1, with aeration to avoid CO2 depletion.

200 mL was mixed with 200 mL of 0.5% thiobarbituric acid (TBA) in 10% TCA. Samples were heated at 95  C for 45 min in a water bath and then cooled to room temperature. Two hundred mL of the mixture were added to each well and the absorbance measured at 532 nm. Malondialdehyde (MDA; SigmaeAldrich) was used as a standard. Concentrations of the pigments chlorophyll a (Chla) and c (Chlc), and fucoxanthin (fx) were measured using a modified version of Seely et al. (1972). A biomass of 200 mg was added to a 1.5 mL tube containing 800 mL of dimethyl sulfoxide (DMSO). After 5 min, samples were centrifuged at 15,000 g for a few seconds to separate biomass from supernatant. Supernatant was diluted in a DMSO: water ratio of 4:1. Pigments were calculated using the equations:

2.3. Transplantation device and field experiments

½Chla ¼ A665 =72:5

E. siliculosus is a small filamentous seaweed that grows up to 30 cm (Charrier et al., 2008); the nature of its morphology and stature would inevitably lead to biomass loss and biofouling (with bacteria and microalgae) if transplanted without being enclosed. Thus, 6 g FW thalli of E. siliculosus were enclosed in 76 mm flat width dialysis tubing (SigmaeAldrich, D9402), cellulose membranes permeable to only small molecules and ions, including metals (De Philippis et al., 2003), and filled with autoclaved seawater. Because of the composition of the tubing and likelihood of it being grazed, it was placed inside transparent 2 L plastic bottles with one hundred 1 mm holes to allow for water exchange (see Fig. 1B). Devices were deployed to field sites and placed 2 m below the lowest tide mark by attaching them to a c. 5 kg rock with nylon fishing line (Fig. 1C). The transplanted material remained in situ for 10 d after which the seaweeds were blotted dried, weighted and immediately frozen in liquid nitrogen, and then stored at 80  C to await analyses.

½Chlc ¼ ðA631 þ A582  0:297A665 Þ=61:8

2.2. Strains and culture

2.4. Metal accumulation Frozen biomass was freeze-dried for 24 h (30e60 mg) and digested with 2 mL of HNO3 in a MARS 6 microwave (Supplementary Table 1). After digestion, the volume of each sample was adjusted to 10 mL with milli-Q water (18 U). The total concentrations of Cu, Cd, Al, Fe and Pb were determined using ICPMS (Thermo Scientific, X Series 2). The choice of metals analysed was based on recently acquired data of high concentrations in
ez sediments and the kelp Lessonia trabeculata from Ventanas (Sa et al., 2012a, 2012b). To validate the data, the digestion protocol was also applied to certified reference material (Ulva lactuca, BCR279) (Supplementary Table 2). 2.5. Measurement of oxidative stress parameters The concentrations of H2O2 were measured according to Sergiev et al. (1997) using a plate reader (VersaMax, Molecular Devices, Sunnyvale, CA, USA). One hundred milligram of frozen biomass were placed in a 1.5 mL tube to which 1 mL of 10% trichloroacetic acid (TCA) was added. Glass beads (3 mm) were added to the tubes and vortexed for 5 min. The homogenate was centrifuged (Sanyo Hawk 15/05) for 10 min at 21,000 g and supernatant was then transferred to a new tube. Fifty mL of supernatant were added to each well, with 150 mL of 50 mM potassium phosphate buffer (pH 7.0) and 100 mL of 1 M KI then added. Absorbance was measured at 390 nm and commercial H2O2 (SigmaeAldrich, G5389) was used as a standard. Levels of lipid peroxidation (LPO) were measured according to Heath and Packer (1968) in a plate reader. The biomass was extracted as for H2O2 measurements. A supernatant volume of

½fx ¼ ðA480  0:722ðA631 þ A582  0:297A665 Þ  0:049A665 Þ  =130

2.6. Activities of antioxidant enzymes Protein extracts for determining antioxidant enzyme activities were prepared as described by Ratkevicius et al. (2003) and, according to that method, proteins were precipitated with ammonium sulphate. Total protein content was determined using bovine serum albumin (BSA) as standard (Ratkevicius et al., 2003). Superoxide dismutase (SOD) activity was determined in a plate reader according to Mishra et al. (1993), with some modifications. Proteins (20 mg) were added to 290 mL of a mixture containing 100 mM potassium phosphate buffer (pH 7.8), 0.1 mM EDTA, 11 mM cytochrome-c, 11 mM xanthine, and 0.002 Units of xanthine oxidase. Activity of SOD was calculated according to Kuthan et al. (1986). Catalase (CAT) activity was determined according to Aebi (1984). The activity was quantified by adding 15 mg of protein extracts to 1 mL of 100 mM potassium phosphate buffer (pH 7) and 16 mM H2O2. The decrease in absorbance was followed at 240 nm for 30 s and the activity was calculated using the extinction coefficient 43.1 M cm1. Ascorbate peroxidase (APX) activity was measured according to Nakano and Asada (1981). Proteins (15 mg) were added to 100 mM potassium phosphate buffer (pH 7), containing 0.5 mM ASC and 16 mM H2O2 (Ratkevicius et al., 2003). The decrease in absorbance at 290 nm was monitored for 30 s and the activity was calculated with the extinction coefficient of ASC (ε ¼ 2.8 mM cm1). The activity of glutathione reductase (GR) was measured in a plate reader according to Sen Gupta et al. (1993). Proteins (50 mg) were added to 290 mL of a solution containing 100 mM potassium phosphate buffer (pH 7), 0.5 mM oxidized glutathione and 0.15 mM NADPH. The decrease in absorbance was followed at 340 nm for 5 min and the activity was quantified with the extinction coefficient of NADPH (ε ¼ 6.22 mM cm1). 2.7. Concentrations of antioxidant compounds The concentrations of reduced ascorbate (ASC) and dehydroascorbate (DHA) were measured according to Benzie and Strain (1999), using a plate reader. A biomass of 300 mg FW was ground to powder in a mortar with liquid nitrogen, and mixed with 1.2 mL 0.1 M HCl. Samples were centrifuged at 21,000 g for 10 min at 4  C. To quantify ASC, 290 mL of tripyridyl triazine (Fe III TPTZ) were


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Table 1 Concentration of metals in the strains of E. siliculosus Es524 and Es147 transplanted to Quintay (pristine site) and Ventanas (polluted site), both located in central Chile, for 10 d. Concentrations of Cu, Fe, and Al are expressed in mmol g1 dry weight (DW) and Cd and Pb are in nmol g1 DW. Different letters represent significant differences for each metal measured (p < 0.05). Errors are ±1 SD, n ¼ 3. Strain Es147 Es147 Es524 Es524

Location Quintay Ventanas Quintay Ventanas

Cu 0.74 8.1 1.12 9.3

Fe ± ± ± ±

b

0.11 0.4 c 0.11 a 0.8 c

0.78 4.4 1.05 5.8

Al ± ± ± ±

added to each well containing 10 mL of extracts, with the assumption that nothing but ascorbate would react. The absorbance at 593 nm was measured immediately after adding Fe III TPTZ. To measure total ascorbate, 500 mL of extracts were incubated in the presence of 5 mL of 100 mM dithiothreitol for 1 h. To stop the reaction, 5 mL (w/v) of N-ethylmaleimide were added. Total ascorbate was measure as for ASC. Concentrations of DHA were calculated by subtracting ASC levels from total ascorbate. L-ASC (SigmaeAldrich) was used as standard. Glutathione in its reduced (GSH) and oxidized (GSSG) forms was measured with modifications to Queval and Noctor (2007). The biomass was extracted as for ascorbate, but in this case the supernatant was neutralized with 5 M K2CO3 to a final pH 6e7. To measure total GSH, 10 mL of supernatant were added to each well, and 290 mL mixture containing 0.1 M NaH2PO4 (pH 7.5) and 6 mM EDTA, 10 mL of 0.34 mM NADPH, 0.4 mM DTNB, and 1 unit of GR were then added. The change in absorbance was monitored at 412 nm for 5 min, and concentrations were calculated using GSH (SigmaeAldrich) as standard. To quantify GSSG, 250 mL of neutralized supernatant were incubated for 20 min with 5 mL of 4vinylpyridine and the mixture was centrifuged at 21,000 g for 5 min at 4  C. Levels of GSSG were measured as for total GSH, and calculated using GSSG (SigmaeAldrich) as standard. Concentrations of phenolic compounds were measured according to Van-Alstyne (1995). One hundred milligram were mixed with 5 mL of 80% methanol in distilled water. Glass beads (c.10, 3 mm) were added and the mixture vortexed at 550 rpm for 24 h at 4  C and then centrifuged at 6000 g at 4  C for 10 min. Supernatant of 12.5 mL was added to 500 mL of 17% Folin-Ciocalteu reagent. After 5 min incubation at room temperature, the solution was made alkaline with 250 mL of 1 M Na2CO3. Samples were placed in a water bath at 50  C and incubated for 30 min. The absorbance was measured at 765 nm using phloroglucinol as standard. 2.8. Statistical analyses The ShapiroeWilk and Bartlett Tests were performed to assess requirements of normality and homogeneity of variances, respectively. To study differences in mean values, one-way ANOVA and post-hoc Tukey test at 95% confidence were performed. Statistical indicators of significant differences were added to figures and tables. 3. Results and discussion 3.1. Metal accumulation The concentrations of Cu, Fe, Al and Pb were significantly higher in E147 and Es524 transplanted to the polluted site of Ventanas than to the pristine location of Quintay (Table 1). At Ventanas, while the accumulation of Cu and Al was not significantly different between strains, the accumulation of Fe and Pb was significantly higher in Es524 than Es147 (Table 1). Differences in the accumulation of metals between strains may reflect cell exclusion capacity

a

0.09 0.4 c 0.11 a 0.8 b

0.4 8.4 0.7 9.6

Pb ± ± ± ±

b

0.05 0.5 c 0.07 a 0.5 c

6.1 52 10.1 78

Cd ± ± ± ±

0.6 3d 1.4 8c

b

a

14.8 6.5 16.6 8.3

± ± ± ±

2.1 1.0 2.7 1.0

a b a b

and cell wall characteristics that can affect competition between metals for binding sites; for example, differences in the conformation of alginic acid which is composed by mannuronic (M) and guluronic acid (G). Changes in M/G ratios have been observed to mediate the availability of binding sites and thus the total metal binding capacity of cell walls (Sinnott, 2007). The concentrations of Cu, Fe, Al and Pb accumulated in Es524 and Es147 at Ventanas were significantly lower than those reported from a passive biomonitoring study at the same location using Lessonia trabeculata, results that are consistent with the shorter period of exposure to the metals. The data confirms Ventanas as a metal-polluted location. In contrast to the above, concentrations of Cd were lower in algae transplanted to Ventanas than to Quintay (Table 1) and are
ez consistent with the results from the study on L. trabeculata (Sa et al., 2012a). Quintay is located in close proximity to one of the most important upwelling hotspots of the south-western Pacific coast (Aiken et al., 2008) and it has been well documented that Cd binds to different biomolecules, and following biological and oceanographic processing is subsequently transported in nutrient
ez et al., 2012b). rich deep waters to coastal areas (see Sa 3.2. Indicators of oxidative damage The concentrations of H2O2 in Es147 were significantly higher than those in Es524 transplanted to the polluted site (Ventanas). There were no significant differences between Es524 and Es147 from the pristine site (Quintay) or between Es524 from Ventanas and Quintay (Fig. 2A). Similarly, the highest levels of lipid peroxidation (LPO) were found in Es147 from Ventanas, while LPO in Es524 from Ventanas and in both strains from Quintay did not differ significantly from one another (Fig. 2B). Thus, Es147 displayed an oxidative stress condition in the highly polluted environment of Ventanas whereas no stress response was observed in Es524, the strain originating from a metal contaminated site. These findings are similar to those obtained in a laboratory study using E. siliculosus strains Es524 and LIA (from a pristine site in N.W. Scotland) exposed up to 2.4 mmol L1 Cu for 10 d; only LIA displayed an oxidative stress condition as evidenced by increases in H2O2 and LPO with increasing Cu exposure (S
aez et al., 2015). Significant decreases in total chlorophyll, Chla and Chlc were found only in strain Es147 transplanted to Ventanas. These results comply with those from the laboratory study on Cu excess described above, with a decrease in chlorophyll concentrations only observed in LIA originating from a pristine site (S
aez et al., 2015). At elevated concentrations Cu can outcompete Fe for uptake, making Fe less available for the synthesis of chlorophyll and thus reducing photosynthetic activity (Patsikka et al., 2002). In addition, Cu can replace Mg in the chlorophyll molecule, reducing photosynthetic performance (Kuepper et al., 2002). However, since there were no differences in pigment concentrations of Es524 transplanted to Quintay and Ventanas, it is more likely that Es147 had a lower capacity to buffer ROS excess than Es524 and consequently experienced greater oxidative damage to chlorophyll

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Fig. 2. H2O2 (A) and lipid peroxidation (B) levels in two strains of E. siliculosus transplanted for 10 d to the pristine site Quintay (white bars), and the metal-polluted location ~ aral, Ventanas (black bars), in central Chile. Strain Es147 is from a low metal-polluted site in Caleta Coloso, northern Chile, and Es524 is from a highly Cu-polluted site in Chan northern Chile. Different letters represent significant difference at 95% confidence interval (p < 0.05). Error bars are ± SD, n ¼ 3.

Fig. 3. Total chlorophyll (A), chlorophyll a (B), chlorophyll c (C), and fucoxanthin (D) concentrations in two strains of E. siliculosus transplanted for 10 d to the pristine site Quintay (white bars), and the metal-polluted location Ventanas (black bars), in central Chile. Strain Es147 is from a low metal-polluted site in Caleta Coloso, northern Chile, and Es524 is from ~ aral, northern Chile. Different letters represent significant difference at 95% confidence interval (p < 0.05). Error bars are ± SD, n ¼ 3. a highly Cu-polluted site in Chan

molecules. This hypothesis is also supported by the LPO results that provide evidence for oxidative damage only in Es147 transplanted to Ventanas. As for fucoxanthin concentrations, there was a significant increase in Es524 transplanted to Ventanas (Fig. 3D), a result that is in agreement with the findings from the in vitro Cu
ez et al., 2015). The carotenoid fucoxanexposure experiment (Sa thin is the main accessory light-harvesting pigment in brown algae. It is very efficient at transferring energy to Chla but may also have a protective role under high irradiance (Evstigne and Paramono, 1974). A strong antioxidant capacity, better even than b-carotene, has been recorded for this pigment (Masashi and Kazuo, 2007), but there is no evidence for any antioxidant role in the metabolism of brown algae. However, the results obtained in this study and from our previous laboratory investigation suggest that fuxoxanthin could be acting as an antioxidant in the chloroplast, providing

protection to chlorophyll from degradation.

3.3. Activities of antioxidant enzymes The activity of SOD has been recognized as the first line of defence against ROS, catalysing the dismutation of
O 2 into H2O2 and O2 (Ken et al., 2005). SOD activity was highest in Es147 from Ventanas (Fig. 4A). Levels in Es524 were lower than in Es147 from Ventanas, but significantly higher than in both strains from Quintay. This information suggests that more SOD is needed in
O 2 stressed Es147 from Ventanas, which would result in greater production of H2O2. CAT and APX are known to cooperate in detoxifying H2O2 in chloroxygenic organisms, such as brown algae (Collen and Davison, 2001; Contreras et al., 2009) and vascular plants (Asada, 1992; Mizuno et al., 1998), including under metal stress. For


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Fig. 4. Activity of antioxidant enzymes superoxide dismutase (SOD) (A), catalase (CAT) (B), ascorbate peroxidase (APX) (C), and glutathione reductase (GR) (D) in two strains of E. siliculosus transplanted for 10 d to the pristine site Quintay (white bars), and the metal-polluted location Ventanas (black bars), in central Chile. Strain Es147 is from a low metal~ aral, northern Chile. Different letters represent significant difference at 95% polluted site in Caleta Coloso, northern Chile, and Es524 is from a highly Cu-polluted site in Chan confidence interval (p < 0.05). Error bars are ± SD, n ¼ 3.

example, Contreras et al. (2009) found that APX and CAT cooperate by increasing their activities in the brown seaweeds Lessonia nigrescens and Scytosiphon lomentaria under Cu stress in vitro, although the extent of the increase differed between the two species. Therefore, to counteract the effects of increased concentrations of H2O2 in Es147 from Ventanas, the activities of CAT and APX might be expected to increase, but this was not observed (Fig. 4B, C). In contrast, CAT and APX activities in Es524, although lower than in Es147 from Ventanas, increased significantly in the material transplanted to Ventanas compared to Quintay, and were sufficiently active to respond to any increases in H2O2 (Fig. 4B,C). Thus, as well as inter-species variation in the activities of APX and CAT in response to metal stress, our results show that activities can be strain (population)-specific. In this particular case, the higher activities of APX and CAT in Es524 when exposed to elevated metal concentrations imply a greater ability to respond to increased H2O2 production compared with Es147 (see Fig. 2A). The enzyme GR is critical in the recycling of glutathione by catalysing the reduction of GSSG to GSH (Noctor et al., 2012). In situ studies on resident seaweed species, such as S. lomentaria (Contreras et al., 2005), and the green seaweed Ulva compressa (Ratkevicius et al., 2003), have usually found lower levels of GR activity in individuals collected from metal contaminated locations than from pristine sites, perhaps indicative of metal-mediated GR cleavage (Schützendübel and Polle, 2002). In contrast, we observed higher levels of GR activity in Es524 from Ventanas than from Quintay, whereas there were no significant changes in Es147 between locations. Our data suggest that under metal-mediated oxidative stress the higher GR activity in Es524 is important for maintaining sufficient GSH, a powerful antioxidant, a metalchelator, and the precursor of the metal-chelating polypeptide, phytochelatin (Noctor et al., 2012).

3.4. Concentrations of antioxidant compounds The behaviour of ascorbate in Es524 and Es147 was similar at the two test sites. There was an almost 20 fold increase in the total ascorbate content of both strains transplanted to Ventanas compared with Quintay (Fig. 5A), indicating that ascorbate was synthesized in response to metal stress. ASC and DHA levels were higher in both strains from Ventanas compared with Quintay, and at Ventanas ASC levels were significantly higher in Es524 than Es147 (Fig. 5B, C). Despite the latter result, the marked increases in DHA in the strains transplanted to Ventanas showed the clearest pattern; DHA levels in both strains from Quintay were around 40% of total ascorbate whereas at Ventanas the values were almost 90%.
ez These results are in general agreement with those reported by Sa et al. (2015); total ascorbate, ASC and DHA increased concomitantly with increasing exposure of up to 2.4 mmol L1 Cu for 10 d in two tolerant strains (Es524 and REP) isolated from polluted locations, while in the less tolerant strain LIA (from a pristine site) ASC levels were significantly lower. Although the results obtained from the field and laboratory experiments are broadly similar, the extent of the increase in DHA differed markedly. Under exposure to Cu in the laboratory DHA concentrations in LIA (non-tolerant) accounted for no more than about 60% of the total ascorbate pool and in the two tolerant strains (Es524 and REP) levels did not exceed 50% of total
ez et al., 2015). These findings imply that the ascorbate (Sa bioavailability of Cu and other metals to E. siliculosus at Ventanas is greater than in our Cu-exposure laboratory experiments and is having a distinct impact on ROS over-production which is apparently counteracted by ascorbate metabolism. Similar results, of marked increases in DHA under metal excess, have been found for L. nigrescens and S. lomentaria (Contreras et al., 2009, 2005) and U. compressa (Mellado et al., 2012; Ratkevicius et al., 2003). Thus, at

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Fig. 5. Total ascorbate (A), reduced ascorbate (ASC) (B), dehydroascorbate (DHA) (C), total glutathione (D), reduced glutathione (GSH) (E), oxidized glutathione (GSSG) (F), and total phenolic compounds (G) concentrations in two strains of E. siliculosus transplanted for 10 d in to pristine site Quintay (white bars), and the metal-polluted location Ventanas (black ~ aral, northern Chile. bars), in central Chile. Strain Es147 is from a low metal-polluted site in Caleta Coloso, northern Chile, and Es524 is from a highly Cu-polluted site in Chan Different letters represent significant difference at 95% confidence interval (p < 0.05). Error bars are ± SD, n ¼ 3.

Ventanas the increased levels of ASC in the more tolerant strain (Es524) are being subsequently oxidized to DHA in order to buffer the metal-mediated ROS production. Synthesis of ASC could either be via the L-galactose pathway, as previously reported by Mellado et al. (2012) in U. compressa or, alternatively, through the HalliwelleAsada Cycle within which GSH is used as a substrate for DHA reduction to ASC by the enzyme dehydroascorbate reductase (DHAR) (Noctor and Foyer, 1998). Since GSH concentrations where higher in Es524 than Es147 from Ventanas (see below), any increase in DHAR activity (not measured) would also have contributed to the higher ASC concentrations in Es524 than Es147 from this location. Patterns in glutathione metabolism were similar to those observed for ascorbate, although not as pronounced. Even though there were higher concentrations of the total glutathione pool in both strains from Ventanas than Quintay, the increase was significantly higher in Es524 with about a 2-fold increase (see Fig. 5D). Similar patterns can be seen for GSH and GSSG (Fig. 5E,F), but the incremental change was greater for GSSG, suggesting an oxidative response. At Ventanas, GSSG levels increased approximately 3-fold in Es524 but only 2-fold in Es147, compared to Quintay. Similar results have been reported for the brown seaweed S. lomentaria (Contreras et al., 2005) and also for several vascular plants exposed to various environmental stressors (Mhamdi et al., 2010; Noctor et al., 2012). As GR catalyses the reduction of GSSG back to GSH, the higher GSH and greater GR activity in Es524 than Es147 from Ventanas can explain, at least in part, better glutathione recycling

in Es524. Furthermore, the increase in GSH in both strains transplanted to Ventanas may have been induced by GSH synthesis through the pathway involving the activities of the enzymes gglutamylcysteine synthetase (g-GCS) and glutathione synthase (GS), as observed in E. siliculosus strains Es524 and REP exposed up to 2.4 mmol L1 Cu (Roncarati et al., 2015). The fact that in both Es524 and Es147 transplanted to Ventanas, GSH levels increased, albeit to a greater extent in the former strain, suggests that glutathione recycling is sufficient to maintain GSH at concentrations capable of counteracting metal-mediated oxidative damage, to some extent. Taking account of our findings on the dynamics of ascorbate and glutathione, together with the changes in the activities of antioxidant enzymes, the information strongly suggests that the HalliwelleAsada (glutathione-ascorbate) cycle is one of the main, if not the most important, antioxidant mechanisms in E. siliculosus and brown seaweeds which allows this ecologically important group macroalgae to thrive in metal contaminated coastal environments. In addition to their antioxidant properties phenolic compounds have been recognized as effective extra- and intra-cellular metal chelators and play an important role in metal-stress metabolism of brown algae (Connan and Stengel, 2011). Concentrations of total phenolic compounds were significantly higher in both strains transplanted to Ventanas than Quintay, with the highest concentrations in Es147 (Fig. 5G). These results are in agreement with the
ez et al. (2015), who found similar increases in total findings of Sa


ez et al. / Environmental Pollution 199 (2015) 130e138 C.A. Sa

phenolic compounds with Cu exposure in three E. siliculosus strains. These results suggest that while phenolic compounds play a role in reactive oxygen metabolism and/or as metal chelators in E. siliculosus under metal exposure, their content is not directly correlated with the pollution histories of the different strains.

3.5. Activation of the antioxidant metabolism and intra-specific tolerance to Cu Of course, other interacting chemical, physical and biological stress factors (e.g. Heinrich et al., 2012; Rijstenbil et al., 2000), in addition to metal exposure, can result in the over-production of ROS and oxidative stress and they may have had some influence on our findings. That notwithstanding, Ventanas and Quintay were selected as suitable study sites because of their similarities in terms of bathymetry, oceanography, geomorphology and marine com
ez et al., 2012a; S
munities (see Sa aez et al., 2012b). Furthermore, the correspondence between the results obtained from previous
ez et al., 2015), laboratory investigations (Roncarati et al., 2015; Sa and those reported here strongly support the view that the intraspecific differences in ROM and the enhanced antioxidant defences observed in strain Es524 are, indeed, in response to metal stress. Other mechanisms also contribute to metal-resistance in E. siliculosus including exclusion from cells by their binding to cell walls and intra-cellular sequestration by metal-chelating compounds such as phytochelatins, as found in our recent laboratory
ez et al., 2015). Thus it is likely that studies (Roncarati et al., 2015; Sa the tolerance of Es524 to metal pollution at Ventanas is achieved through a combination of efficient cell exclusion, antioxidant defences and enhanced production of metal-chelators such as phytochelatins. Future field experiments will address this hypothesis.

3.6. Conclusions The deployment of E. siliculosus within dialysis tubing to field sites for 10 d proved to be very effective, with no loss of material and very little biofouling of either the tubing or the protective bottles. The results obtained from the study offer a reliable representation of the metal bioavailability at the two locations, being in general agreement with the available published data that confirms
lez Ventanas to be the more metal-contaminated location (Gonza
ez et al., 2012a, 2012b). These reasons, together et al., 2008; Sa with its simplicity and cost-effectiveness, make this novel device and the approach highly suitable for incorporating into biomonitoring programmes for assessing chemical pollution in coastal waters and estuaries. The methodology would lend itself to other filamentous and frondose seaweed species and to the microscopic stages (e.g. gametophytes, germlings) of morphologically more complex ones. In this study we were able to establish that the responses recorded in a serious of laboratory studies reflect those occurring in nature. The congruence between field and laboratory results strongly imply that the different responses of Es524 and Es147 are mediated by metal stress and confirm Es524, isolated from a highly metal-impacted location, as the more tolerant strain. Therefore, for the first time employing field transplantation experiments, we provide unambiguous evidence for inter-population variation in metal-tolerance in brown seaweeds. In the case of E. siliculosus strain Es524 the mechanisms include a strong antioxidant capacity which is associated with the synthesis of the antioxidants GSH and ASC, and increased activities of the antioxidant enzymes SOD, APX, CAT, and GR.

137

Acknowledgements This study was funded by the Santander Postgraduate Mobility Support Scholarship 2011e12, awarded at Plymouth University to C.
ez. We thank financial support for PhD studies to C. A. Sa
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