Antioxidant Efficiency of Platynereis Spp.(Annelida, Nereididae) Under Different Ph Conditions at a Vent's System

Hindawi Journal of Marine Biology Volume 2019, Article ID 8415916, 9 pages https://doi.org/10.1155/2019/8415916

Research Article Antioxidant Efficiency of Platynereis spp. (Annelida, Nereididae) under Different pH Conditions at a CO2 Vent’s System

Giulia Valvassori ,1 Maura Benedetti,2 Francesco Regoli,2 and Maria Cristina Gambi1

1 Stazione Zoologica Anton Dohrn Napoli, Dept. of Integrative Marine Ecology, Villa Dohrn-Benthic Ecology Center, 80077 Ischia, Napoli, Italy 2UniversitaPolitecnicadelleMarche,Dept.DiSVA(DipartimentodiScienzedellaVitaedell’Ambiente),` Laboratory of Ecotoxicology and Environmental Chemistry, 60131 Ancona, Italy

Correspondence should be addressed to Giulia Valvassori; [email protected]

Received 29 June 2018; Revised 19 December 2018; Accepted 2 January 2019; Published 20 January 2019

Academic Editor: Horst Felbeck

Copyright © 2019 Giulia Valvassori et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Marine organisms are exposed to a pH decrease and to alteration of carbonate chemistry due to ocean acidifcation (OA) that can represent a source of oxidative stress which can signifcantly afect their antioxidant defence systems efciency. Te polychaetes Platynereis dumerilii and P. massiliensis (Nereididae) are key species of the benthic community to investigate the efect of OA due to their physiological and ecological characteristics that enable them to persist even in naturally acidifed CO2 vent systems. Previous studies have documented the ability of these species to adapt to OA afer short- and long-term translocation experiments, but no one has ever evaluated the basal antioxidant system efciency comparing populations permanently living in habitat characterized by diferent pH conditions (acidifed vs. control). Here, individuals of both Platynereis species, sampled from a natural CO2 vent system and from a nonventing “control” site in three diferent periods (April 2016, October 2016, and February 2017), were compared highlighting signals which suggested the ability of both species to acclimatize to high pCO2–low pH with slight seasonal variations of their antioxidant efciency and the absence of disturbances of the oxidative status of Platynereis spp. tissues.

1. Introduction (ROS) additional production. ROS, endogenous and highly reactive oxygen-bearing molecules, are commonly produced World climatic alterations are mainly driven by atmospheric at low concentrations during several natural cellular pathways CO2 partial pressure (pCO2) increase, as a consequence of the of aerobic metabolism, and under basal and stable conditions anthropogenic activity, which is predicted to reach 800 ppm their adverse efects are prevented by antioxidant defence by the end of the current century [1–5]. Tis change at the systems (i.e., low molecular weight scavengers and enzymes). atmospheric level also afects the ocean surface through the In stressful conditions, this balance may be altered leading phenomenon of ocean acidifcation (OA): an increase of the to uncontrolled ROS formation that translates into cellular dissolved CO2, alteration of seawater carbonate chemistry, oxidative damage against biological macromolecules includ- and the consequent reduction of the ocean pH. Ocean surface ing lipids, proteins, and DNA, impairing normal cellular pH has fallen by about 0.1 units since the beginning of the functions. Tis unbalance in favour of oxidants is termed industrial era and is expected to further decrease by 0.3-0.5 “oxidative stress”. In the last years, scientifc literature has units by the end of the current century [1, 4, 5]. Marine organ- provided evidence that global climate change, especially OA, isms are continuously exposed to a range of environmental afects antioxidant systems efciency of several marine organ- parameters, such as pH, salinity, and temperature, varying isms [6–20]. Te biological efects of low pH–high pCO2 have over temporal and spatial scales, which may represent a been investigated through not only laboratory/mesocosm source of oxidative stress that entails Reactive Oxygen Species experiments, but also studying natural volcanic CO2 vent 2 Journal of Marine Biology systems which occur in diferent parts of the world (e.g., experiments on both species also highlighted a diferenti- [21–28]). Such “natural laboratories” have so far provided ation on the expression of some target genes involved in environmentally realistic overviews of the conditions with the oxidative metabolism as a result of the exposure to which marine organisms will interface according to the near- diferent pH conditions [48, 49]. Specimens of Platynereis future OA predictions [5, 29] and represent an important tool dumerilii from a control site near the vents, Sant’Anna, to detect information about putatively tolerant species to OA showed signifcant lower levels of NADH dehydrogenase and their capability to modulate the antioxidant system in mRNA expression compared to P. cfr massiliensis from response to pH variations. Despite the growing interest of Castello acidifed sites confrming, as already stated by Calosi the scientifc community in this research feld, there is still et al. [24], that living under acidifed conditions entails a little knowledge about how the acidifcation processes afect higher energetic consumption and metabolic rate [48]. In antioxidant defence systems of the benthic biota. line with Calosi et al. [24], translocation experiment did not Within benthic community, polychaetes represent a key show signifcant efect for this gene expression in Platynereis group in marine habitat and are ofen used as bioindicators dumerilii but, in contrast, a signifcant downregulation of in monitoring programs for their high sensitivity to metal P. cfr massiliensis NADH dehydrogenase from low pH to exposure [30–33] and anthropogenic pressure [34, 35]. Due to control conditions was observed indicating a reduction in their physiological and ecological characteristics, polychaetes the oxidative metabolism of this species [48]. Diferently to are among the most abundant invertebrates under low pH what was previously asserted by Lucey et al. [46], recent conditions, such as along the naturally acidifed CO2 vent phylogenetic analysis carried out in the frame of a PhD system of the Castello at the Ischia Island [22, 36–39], and thesis on Platynereis spp.[50],samplescollectedinthesouth- various studies investigated the role of antioxidant systems acidifed areas of Castello Aragonese and the control zone of in response to low pH–high pCO2 [12, 16–18, 24, 40, 41]. Sant’Anna rocks highlighted a less evident spatial segregation Te polychaete Platynereis dumerilii (Audouin and Milne- of the two Platynereis species between the two sites. For this Edwards, 1834) (Nereididae) represents a key species: it reason, it was not possible to consider the Castello vent area showed high tolerance to environmental stress [42, 43], as an exclusive domain zone for P. massiliensis,aswellasthe including low pH [38], and for this reason it was employed as control area of Sant’Anna for P. dume r i lii . model organism in two recent in situ transplant experiments, Considering the preliminary results on antioxidant ef- carried out to evaluate the efects of OA on the oxidative ciency of putative P. dume r i lii and the presence of both species sensitivity of populations living inside and outside the vent in control and acidifed sites, the aim of this study was to area of Ischia [12, 24]. Te short-term translocation (only 5 provide new insights about the basal levels of the antioxidant days) displayed a true local adaptation to low pH conditions, system in Platynereis spp., comparing populations living at with an exclusive genotype apparently restricted to the diferent pH conditions (acidifed vs. control). Tis compar- acidifed areas of Castello Aragonese (Ischia) characterized ison between populations was carried out in three diferent by higher metabolic rate, measured as oxygen consumption periods: April 2016, October 2016, and February 2017, in [24]. Te genotype of the acidifed areas was later identifed relation to diferent temperature conditions. Te oxidative as the only known sibling species of Platynereis dumerilii, P. efects of diferent pH levels were evaluated thorough analysis massiliensis (Moquin-Tandon, 1869): morphologically indis- of single antioxidant activities, such as catalase (CAT), glu- tinguishable species in its adult (nonreproductive) stage, but tathione S-transferases (GST), glutathione reductase (GR), characterized by a completely diferent reproductive biology and Se-dependent and Se-independent glutathione peroxi- [44–46]. A recently published study that combined together dases (GPx), that can be very sensitive in revealing a prooxi- genetic and reproductive biology analyses revealed that dant condition [51] and thorough their integration with total both Platynereis species actually represented two diferent oxyradical scavenging capacity (TOSC), which quantify the complexes of siblings [47]. Based on preliminary genetic capability of Platynereis spp. to neutralize diferent forms ∙ results, the Castello vent site of Ischia appeared dominated of oxyradicals including peroxyl radicals (ROO ), hydroxyl ∙ by the brooding P. massiliensis sibling, while the control radicals (HO ), and peroxynitrite (HOONO) [52]. site by the broadcasting species P. dume r i lii [24, 46, 47]. In the long-term translocation (30 days) the antioxidant sen- 2. Materials and Methods sitivity of diferent polychaete species (Platynereis dumerilii, Polyophthalmus pictus, and Syllis prolifera) and their antioxi- 2.1. Study Areas, Sample Collection, and Processing. Te study dant capacity to counteract oxyradicals formation in control wasconductedattheIschiaisland(GulfofNaples,Italy), and low pH conditions was evaluated, highlighting as the a volcanic island well known for the presence of numerous population of Platynereis originating from the vent showed submarine CO2 vent systems [29, 53, 54], including the area ∘ higher constitutive antioxidant efciency [12], which may of Castello Aragonese on the north-eastern side (40 43.84 N, ∘ allow them to cope with short-term and chronic exposure 13 57.08 E) as the frst vent system studied in the world [29]. to higher oxidative pressure without further enhancement of Gas bubbles, composed by 90-95% CO2,3-6%N2, 0.6-0.8% antioxidant defences [12]. From these results, a hypothesis O2,0.2-0.8%CH4, with no sulphur, are released at ambient 6 −1 of long-term adaptation of the Platynereis vent-inhabiting seawater temperature at about 1.4 x 10 ld [21]. Te salinity −1 population emerged, suggesting the need of this species for of the water (38) and total alkalinity (2.5 mequiv Kg ) greater antioxidant protection in conditions of chronic oxida- are relatively uniform [21, 38]. Gas emissions, which occur tive exposure to low pH–high pCO2 [12]. Recent laboratory between 0.5 and 3.0 m depth, create a gradient of pH on both Journal of Marine Biology 3

2.2. Analyses of Antioxidants and Total Oxyradical Scaveng- ing Capacity. For the analysis of the antioxidant enzyme N2 N1 N3 activities, pools were homogenized (1:10 w:v) in 100 mM of potassium phosphate bufer (pH 7.5) containing NaCl (1.5%), Castello −1 S3 0.1 mg mL phenylmethylsulphonyl fuoride (PMSF), 0.1 mg Aragonese −1 −1 S2 mL bacitracin and 0.008 TIU mL aprotinin as protease S1 inhibitors. Afer centrifuging at 100,000 x g for 70 min at ∘ 4 C, supernatants were collected and used for the subse- quent analyses. Enzymatic activity measurements including catalase (CAT), glutathione S-transferases (GST), glutathione reductase (GR), and glutathione peroxidases (GPx) were S.Anna carried out using a Varian (Model Cary 3) spectrophotometer ∘ 0 100 at the constant temperature of 18 C according to Bocchetti Meters et al. [30]. CAT activity was determined by the decrease in � −1 −1 Figure 1: Ischia island sampling sites: Castello Aragonese with absorbance at 240 nm ( =0.04mM cm )duetoH2O2 acidifed (S3, S2, N3, N2) and control (S1, N1) stations and the consumption (12 mM) in 100 mM K-phosphate bufer (pH control site at Sant’Anna rocks. 7.0). GST activity was quantifed at 340 nm using 1-chloro-2,4 −1 −1 dinitrobenzene (CDNB) as substrate (� =9.6mM cm ). Te assay conditions were 100 mM potassium phosphate bufer(pH6.5),1.5mMCDNB,and1.5mMGSH.GRactivity, the north and south sides of the Castello islet. According to also known as glutathione-disulphide reductase (GSR), was Kroeker et al. [22], three diferent pH zones can be identifed measured spectrophotometrically at 340 nm following the alongarockyreefofapproximately150minlengthoneach oxidation of NADPH during the reduction of GSSG (extinc- −1 −1 side of the islet: a control area with normal pH conditions tion coefcient, � =6.22mM cm ). Te assay was carried and no venting activity (N1 and S1), an intermediate area with outin100mMpotassiumphosphatebufer(pH7.0),1mM moderate vent activity and low pH conditions (N2 and S2), GSSG and 60 �M NADPH. Te activity of Se-dependent and and a high venting activity area characterized by extreme low Se-independent GPx forms was determined in two enzymatic pH conditions (N3 and S3) (Figure 1). Te acidifed S2–S3 assays in which GSSG is converted to the reduced form GSH. stations where Platynereis collection was performed showed Te consumption of NADPH was quantifed as decrease of −1 −1 a wide range of pH variability as reported by Ricevuto et al. absorbance at 340 nm (� =6.22mM cm )in100mMK- [38, Supplement material]. Te control site, called Sant’Anna phosphate bufer pH 7.5, 1 mM EDTA, 1 mM dithiothreitol rocks, characterized by a very stable pH value, which ranged (DTT), 2 mM GSH, 1-unit glutathione reductase, 0.24 mM around a mean of 8.01, is located within the Cartaromana NADPH, and 0.8 mM cumene hydroperoxide as substrate. Bay, approximately 600 m from the south side of Castello For the total oxyradical scavenging capacity (TOSC) Aragonese [12] (Figure 1). analysis, polychaetes were homogenized following the same Platynereis spp. samples were collected in the south- −1 protocol reported above, with 0.5 �gmL pepstatin as acidifed sites of Castello (named as S3 and S2 in previous additional protease inhibitor and without phenylmethyl- papers e.g., [36], or as low pH and extreme low pH in [22]) sulphonyl fuoride (PMSF). Tis assay was based on the and in the normal/control pH area of Sant’Anna rocks. Sam- capability of cellular antioxidants to reduce the oxidation of plings were carried out in four diferent periods: April 2016, �-keto-�-methiolbutyric acid (KMBA), and the consequent October 2016, and February 2017 (mean monthly seawater ∘ ∘ ∘ formation of ethylene gas, in presence of artifcially generated temperatures 16.5 C, 21.9 C, and 14.6 C, respectively). Worms oxyradicals. Te ethylene formation was monitored at 12 werecollectedineachsamplingsiteandperiodbysampling min time intervals by gas-chromatographic analyses and the local macroalgae belonging to the species Halopteris scoparia, TOSC values were calculated from the equation: TOSC = Jania rubens, Dictyota spp., and Cladophora spp. where these 100 – (∫ SA/∫ CA x 100), where ∫ SA and ∫ CA were the polychaetes live associated. Macroalgal thalli were collected integrated areas calculated under the kinetic curve produced in cotton fabric bags by snorkelling and SCUBA diving at during the reaction course for sample (SA) and control (CA) 0.5–2 m depth. Afer collection, samples were transported reactions, respectively [52]. to the Villa Dohrn-Benthic Ecology Center (approx. 4 km In order to obtain the specifc antioxidant activity and from the Castello area) inside cool boxes within one hour. TOSC values, data were normalized with the relative protein Once in the lab, algal thalli were sorted and the Platynereis concentration according to Lowry method [55] by using spp. species was identifed thanks to the typical sinuous Bovine Serum Albumin (BSA) as standard. swimming movement, immediately transferred into separated 1.5 ml microcentrifuge tubes (pooling approximately 5-10 individuals per Eppendorf, according to the body mass 2.3. Statistical Analysis of Data. Permutational multivariate of the collected samples), frozen, and temporarily stored at - analysis of variance (PERMANOVA) and pairwise non- ∘ 80 C until the transport to the Laboratory of Ecotoxicology parametric tests with square root transformation, Euclidean and Environmental Chemistry of Ancona (Italy) for the distance, and 9,999 number of permutations were conducted antioxidant analyses. to test the diferences (� = 0.05). “Population origin” and 4 Journal of Marine Biology

Table 1: Sampling periods’ variations of biomarkers (mean values ± standard deviations, �=5)inPlatynereis spp. population from Sant’Anna (SA, normal pH conditions) and Castello Aragonese (CA, acidifed pH conditions).

April October February Period x population Mean s.d. Mean s.d. Mean s.d. SA 52.91a 15.88 35.52b∗ 1.85 24.05c∗ 6.91 CAT CA 48.63 20.33 44.82 1.95 45.66 9.78 SA 58.58a 24.73 29.62b 4.63 30.44b 8.58 GST CA 37.75ab 12.10 34.24a 5.05 26.59b 5.62 SA 54.53a∗ 25.45 30.43a 6.94 6.98b∗ 3.47 GR p < 0.001 CA 21.68a 2.06 32.11b 5.94 22.67ab 5.15 SA 13.91 6.20 26.29 13.67 20.32 5.39 GPx CA 16.3a 3.01 23.29b 4.64 18.05ab 3.97 SA 1044.88 359.44 904.99 203.17 774.35 146.88 ROO∙ CA 918.83 331.44 988.68 312.71 946.79 270.06 SA 654.36 240.36 581.23 157.36 647.05 180.70 HO∙ CA 640.78ab 181.19 748.18a 186.85 446.85b 89.39 SA 500.79a 127.41 786.5b∗ 101.88 336.42c∗ 37.99 HOONO p < 0.01 CA 451.32 142.57 522.71 134.84 507.98 139.86 CAT: catalase (�mol/min/mg prt); GST: glutathione S-transferases (nmol/min/mg prt); GR: glutathione reductase (nmol/min/mg prt); GPx: glutathione peroxidases (nmol/min/mg prt); ROO∙,HO∙, and HOONO: TOSC toward peroxyl, hydroxyl radicals, and peroxynitrite (UTosc/mg prt). Te p values are given for the interaction “period x population” when it resulted in statistical signifcance (PERMANOVA); diferent letters indicate signifcant diferences among sampling periods (p < 0.05), while asterisks indicate signifcant diferences between Sant’Anna and Castello Aragonese Platynereis spp. populations (p < 0.05) (PERMANOVA pairwise post hoc comparison).

“sampling period” were considered as fxed factors with two TOSC efciency measured in October (HOONO = 786.5 and three levels, respectively (sites: Castello and Sant’Anna; UTosc/mg prt) and the lowest one in February (336.42 periods: April 2016, October 2016, February 2017), to test UTosc/mg prt). Concerning other antioxidant parameters, in the response temporal variation between population. Mul- worms sampled from Sant’Anna, CAT exhibited the highest tivariate principal component analysis (PCA) was applied activity in April, followed by a signifcant decrease over to visualize the relationships among the diferent popu- the examined periods; on the contrary, Castello specimens lations/sampling periods and all statistical analyses were showed constant values with some diferences (p < 0.05) performed using PRIMER/PERMANOVA v 6 [56]. between specimens in October and February (Figure 2). Simi- lartrend,withthehighestenzymaticactivityinApril,wasalso 3. Results observed for the GST in both populations. On the contrary, the activity of Se-dependent and Se-independent GPx was Results are showed in both Table 1 and Figure 2; the activity lowerinAprilandincreasedinOctoberandFebruaryin organisms sampled from both sites. TOSC values toward of GR signifcantly difered between the two Platynereis ∙ ∙ spp. populations in April and February (pairwise compar- ROO did not show signifcant diferences, while against HO ison p < 0.05, see asterisks Table 1). Sant’Anna population limitedvariationswereobservedinorganismssampledfrom showed signifcant diferences between April–October and Castello with a signifcant decrease in February. April–February (pairwise comparison p < 0.05, see letters PCA analysis provided a two-dimensional pattern Table 1). Te highest enzyme activity was recorded in April explaining 82.5% of the total variance (56.2% and 26.3% in (GR = 54.53 nmol/min/mg prt) and the lowest one in the frst and second axes, respectively) (Figure 3). However, February (GR = 6.98 nmol/min/mg prt). On the contrary, despite some diferences occurred mainly for single GR activity was higher in October in organisms sampled antioxidants activity between populations and periods, no from Castello with values comparable to those observed clear groups’ separation occurred (Figure 3). in Sant’Anna population (SA = 30.43 nmol/min/mg prt; CA = 32.11 nmol/min/mg prt), while constant values were 4. Discussion observed for this population in the other months. Concern- ing the capability to neutralize HOONO, the Platynereis spp. Tis study represents the frst attempt of a background populations showed signifcant diferences in October and analysis of the antioxidant parameters of Platynereis spp. February between the investigated sites (Figure 2). While (putative diferent sibling species) living in diferent pH the total oxyradical scavenging capacity toward HOONO in conditions (normal and acidifed conditions). Specimens organismsfromCastellowasconstantoverdiferentsampling morphologically identifed as Platynereis dumerilii were periods, conversely, more marked variation was observed for already employed as model organisms for some transplant Platynereis specimens from Sant’Anna site, with the highest experiments to investigate the prooxidant efect of ocean Journal of Marine Biology 5

CAT GST 80 90 70 80 60 70 60 50 50 40 40 30 30 20 20 nmol / min / mg protein / min mg nmol mol / min / mg protein / min mg  mol 10 10 0 0 April 2016 October 2016 February 2017 April 2016 October 2016 February 2017

GR GPx 90 45 a∗ 80 40 70 35 60 30 50 25 ab 40 20 30 a ab 15 20 10 b∗ nmol / min / mg protein / min mg nmol 10 protein / min mg nmol 5 0 0 April 2016 October 2016 February 2017 April 2016 October 2016 February 2017

ROO∙ HO∙ 1600 1000 1400 900 800 1200 700 1000 600 800 500 600 400 300 400 200

TOSC units / mg protein / mg units TOSC 200 protein / mg units TOSC 100 0 0 April 2016 October 2016 February 2017 April 2016 October 2016 February 2017

ONOOH 1000 b∗ 900 800 700 a 600 500 c∗ 400 300 200

TOSC units / mg protein / mg units TOSC 100 0 April 2016 October 2016 February 2017

Sant'Anna Castello Aragonese

Figure 2: Basal antioxidant enzyme activities (CAT, GST, GR, GPx) and total oxyradical scavenging capacity (TOSC) toward peroxyl ∙ ∙ radicals (ROO ), hydroxyl radicals (HO ), and peroxynitrite (ONOOH) in Sant’Anna (white) and Castello Aragonese (black) Platynereis −1 −1 −1 −1 spp. populations. Values are expressed as �mol min mg protein for CAT; nmol min mg protein for GST, GR and GPx; TOSC units/mg ∙ ∙ protein for ROO ,HO, and ONOOH. Letters indicate signifcant diferences among sampling periods (� < 0.05), while asterisks indicate signifcant diferences between Sant’Anna and Castello Aragonese Platynereis spp. populations (� < 0.05) (PERMANOVA pairwise post hoc comparison). 6 Journal of Marine Biology

5 specimens morphologically identifed as Platynereis dumerilii displayed insights of long-term adaptation of the vent population to greater prooxidant challenge [12]. Conversely to our fndings, Ricevuto et al. [12] observed that Platynereis ventspecimensshowedmoreelevatedbasalantioxidant 0 ∙ PC 2 efciency toward ROO and HOONO when compared to the population collected in control pH conditions, suggesting the need of a greater antioxidant protection in conditions of chronic oxidative exposure [12]. A diferent response to −5 pH conditions was also highlighted by Wage¨ et al. [48] in −10 −5 0 5 10 whose study P. cfr massiliensis from acidifed sites showed PC1 a marked upregulation of gene expression involved in the Figure 3: Separation of Platynereis spp. populations (white circles energy metabolism compared to P. dume r i lii from control = Sant’Anna; black squares = Castello Aragonese) and periods of site. Tis inconsistency with our results might be due to a sampling obtained by principal component analysis (PCA) of mean mixing of the two species (or complexes) in the studied areas, biological responses. Castello Aragonese and Sant’Anna rocks, only 600 meters away from each other. Te two species would therefore be separated exclusively by a chemical barrier (pH) for which nonsignifcant efects were detected at least in P. dume r i lii acidifcation [12, 24, 48, 49], but the basal level efciency of [48]. the antioxidant defence systems in terms of species sensi- Te PCA analysis suggested that the antioxidant response tivities and seasonality of populations submitted to natural of Sant’Anna population was more diferentiated when com- acidifed and normal pH conditions was never evaluated. pared with specimens collected in the acidifed areas of Our overall results indicated that the two populations Castello, without a clear trend in the diferent periods showed few diferences, probably based on seasonal-related (Figure 3). Both statistical analyses supported that the two conditions in their habitat, in particular for glutathione populations seemed to have diferent temporal-related trends reductase activity and the total oxyradical capability to of the antioxidant defence systems, even if the lowest mean counteract HOONO radical. GR catalyses the reduction values of antioxidant capacity were mainly recorded in the of glutathione disulphide (GSSG) to the active form of period characterized by the lowest water temperature (Febru- glutathione (GSH) which is a fundamental molecule for ary). Tis phenomenon was most evident in the Sant’Anna preventing oxidative stress and maintaining the reduced envi- population, which showed a marked decrease of antioxidant ronment of cell, using NADPH as cofactor. Te GR activity of activities of CAT, GST, and GR and a lower scavenging ∙ specimens sampled from control site (Sant’Anna population) capacity toward ROO and HOONO, in February. Te showed the highest activity in April and a rapid decrease until antioxidant defence systems’ efciency of marine organisms February. Conversely, the Castello vent population showed can be infuenced by several environmental factors, such similar trend and values during the whole year, suggesting as annual fuctuations in solar irradiance, changes in water more stable environmental conditions during the examined temperature, fuctuating oxygen concentration, and exposure periods. Similar considerations were also supposed for TOSC to chemical pollutants [58–63]. Te lowest temperature of the toward HOONO, which showed for specimens collected in winter season could entail lower prooxidant pressure and, as a Sant’Anna a maximum capability to counteract radical species consequence, the need of a decreased antioxidant efciency, in October and a minimum one in February, while the as reported for the European eel, Anguilla anguilla,[64],in vent population displayed constant activity during the whole order to counteract the increase of environmental ROS for- sampling period. mation during the summer. High temperature increased ROS Based on limited variations, our fndings did not show production and the consequent enhancement of antioxidant a clear correlation between pH conditions and alteration of enzymes’ activity also in the mussel species Mytilus coruscus the oxidative status in Platynereis spp. populations, which [57]. was instead already observed in previous ocean acidifcation In this study, Platynereis spp. population from control pH experiments with several invertebrate species [6, 8, 10–12, conditions displayed a higher temporal variability, showing 14–16, 41, 48, 49, 57]. An in situ transplant experiment of the need to modulate the redox response to keep the oxidative 30 days into naturally acidifed conditions carried out with stress level of the tissues under control during diferent the fan worm Sabella spallanzanii (Sabellidae) highlighted a periods of the year. Conversely, the overall ability of the vent signifcant decrease of enzymatic activities of CAT and GPx population to maintain stable levels of antioxidant defences, and the impairment of the overall capability to neutralize regardless the period of the year and seasonal-related trends, ∙ hydroxyl radicals (HO ) [41]. Te efect of pH decrease was suggested that natural enhancement of environmental proox- also investigated in the polychaete Diopatra neapolitana;afer idant conditions was balanced with slight changes of indi- 28 days of exposure to low pH levels higher enzymatic activity vidual antioxidants. Te diferent native pH conditions, over (CAT, SOD, GSTs) and oxidative alterations were recorded three sampling periods, were not translated into signifcant [16]. Te results of TOSC assay performed afer an in situ diferences between populations in the other antioxidant reciprocal transplant experiment in the Castello vents on biomarkers analysed, and the similar antioxidant responses Journal of Marine Biology 7 highlighted between the two populations confrmed the high [2] K. Caldeira and M. E. Wickett, “Ocean model predictions of tolerance of these species (or complex of sibling species, chemistry changes from carbon dioxide emissions to the atmo- [47]). Te long-term exposure to moderately elevated pCO2 sphere and ocean,” Journal of Geophysical Research: Oceans,vol. conditions, such as those expected in global climate change 110,no.9,pp.1–12,2005. scenarios at the end of this century [1], could minimally [3]J.C.Orr,V.J.Fabry,O.Aumontetal.,“Anthropogenicocean afectthecellularredoxstatus,asalreadyobservedintwo acidifcation over the twenty-frst century and its impact on marine bivalve species, Crassostrea virginica and Mercenaria calcifying organisms,” Nature,vol.437,no.7059,pp.681–686, mercenaria [9]. 2005. [4] J. Raven, K. Caldeira, H. Elderfeld et al., “Ocean acidifcation due to increasing atmospheric carbon dioxide,” Te Royal 5. Conclusions Society Policy Document 12.05, Clyvedon Press, Cardif, UK, 2005. In conclusion, this study provided the frst baseline for a [5] IPCC, Climate Change 2014: Synthesis Report. 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