Isolation-hypoxia and re-oxygenation of the pallial cavity of female Crepipatella dilatata during estuarine salinity changes requires increased glyoxylase activity and antioxidant metabolism to avoid oxidative damage to female tissues and developing embryos

Marine Environmental Research 119 (2016) 59e71

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Isolation-hypoxia and re-oxygenation of the pallial cavity of female Crepipatella dilatata during estuarine salinity changes requires increased glyoxylase activity and antioxidant metabolism to avoid oxidative damage to female tissues and developing embryos Víctor Cubillos a, b, *, Oscar Chaparro a, Cristian Segura a, Jaime Montory a, Edgardo Cruces c, David Burritt d
gicas, Universidad Austral de Chile, Valdivia, Chile Instituto de Ciencias Marinas y Limnolo
ticos de Calfuco, Universidad Austral de Chile, Valdivia, Chile Laboratorio Costero de Recursos Acua c Center for Development of Nanoscience and Nanotechnology (CEDENNA), Universidad de Santiago de Chile, Santiago, Chile d Department of Botany, University of Otago, Dunedin, New Zealand a

b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 22 February 2016 Received in revised form 3 May 2016 Accepted 7 May 2016 Available online 9 May 2016

The estuarine slipper limpet Crepipatella dilatata is a gastropod that can survive prolonged periods of low salinities (< 24 PSU) caused by tidal changes and/or prolonged periods of rain. During low salinity events, C. dilatata can isolate its body from the outside environment, by sealing its shell against the substrate on which it grows. Prolonged isolation periods from the surrounding environment can greatly lower available oxygen levels inside of the pallial cavity, impacting on the physiology of both females and their incubated encapsulated embryos. When salinity levels return to normal, isolation is terminated and the inflow of seawater results in re-oxygenation. In this study we show that when re-oxygenation of the pallial cavity takes place, oxidative damage, in the form of increased levels of lipid peroxides and protein carbonyls, occurs in both maternal tissues and in incubated embryos. To avoid terminal oxidative damage both females and their embryos increase their levels of the glyoxalase pathway enzymes (GLX-I and GLXII) and general antioxidant metabolism (SOD, CAT, GR, GPOX and GST). As a result the levels of oxidative damage decline to basal levels within 24 h of reoxygenation. Thus the combination of isolation, a behavioural strategy, combined with encapsulation of embryos and a capacity to up regulate relatively rapidly the glyoxylase pathway and general antioxidant metabolism, play major roles in facilitating the survival of C. dilatata in the small estuaries of Southern Chile. © 2016 Published by Elsevier Ltd.

Keywords: Brooding Encapsulated embryos Re-oxygenation Oxidative damage Antioxidants

1. Introduction Estuarine areas are considered to be among the most stressful marine environments, due to periodical fluctuations in the physicochemical conditions of the water column as a consequence of tidal changes (Chaparro et al., 2008c; Huang et al., 2003). Salinity is one of the most important environmental variables in estuaries (Kinne, 1966; Roast et al., 1999), and regulates many of the physiological and behavioural responses of estuarine organisms (Chaparro et al.,
lez, 2008b; Marsden, 2004; Navarro, 1988; Navarro and Gonza 1998). Changes in salinity can cause osmotic stress and also

* Corresponding author. E-mail address: [email protected] (V. Cubillos). http://dx.doi.org/10.1016/j.marenvres.2016.05.008 0141-1136/© 2016 Published by Elsevier Ltd.

impact feeding behaviour, growth rates and the survival of estuarine organisms (Castagna and Chanley, 1973; Chaparro et al., 2008b; Kinne, 1966; Mcgaw et al., 1999; Pechenik et al., 2000). During tidal changes mobile estuarine species can move from low to high salinity areas to reduce osmotic damage (McGaw and Naylor, 1992). In contrast, sessile species have evolved different strategies, combining behavioural and physiological responses, in addition to the development of specific anatomical structures, to ameliorate fluctuations in salinity during low tides or periods of heavy rain (McFaruume, 1980; Pardo et al., 2011). To compensate for their lack of movement, during periods of environmental low salinity, sessile or semi-sessile molluscs that inhabit estuarine ecosystems can close their valves or clamp their shells tightly over a substrate, thus isolating themselves from the surrounding

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environment (Chaparro et al., 2008b; Montory et al., 2009; Segura et al., 2015). Prolonged periods of isolation of the pallial cavity from the surrounding environment, can lead to detrimental conditions within the pallial cavity, including reduced pH, elevated ammonia levels and reduced oxygen levels (Chaparro et al., 2009; Segura et al., 2015), which can significantly impact metabolic rates, gene expression, the activities of ion pumps and finally trigger proteolysis (Hochachka, 1997; Letendre et al., 2012). Under hypometabolic conditions, reactive oxygen species (ROS) such as the superoxide anion radical (O2
), hydrogen peroxide (H2O2), singlet oxygen (1O2) and hydroxyl radical (HO
) can be generated in marine organisms (Chandel and Schumacker, 2000; Lesser, 2006). If the rate of ROS production exceeds the rate of removal, ROS accumulate in cells and can oxidise cellular macromolecules resulting in oxidative damage to lipids, proteins and DNA (Ahmad, 1995; Bergamini et al., 2004; Halliwell, 1987; Halliwell and Chirico, 1993; Hermes-Lima, 2005; Lesser, 2006). Although tidal cycles can generate a stressful microenvironment in the pallial cavity of some molluscs (Chaparro et al., 2009; Montory et al., 2009), estuarine species have adapted though behavioural and physiological responses to cope with periodical variations in the salinity levels (Chaparro et al., 2009; Montory et al., 2009; Segura et al., 2010). While it is likely that changes in antioxidant metabolism would be required to cope with the increase in ROS generation under the above conditions, little is known about these changes. In animals, ROS are detoxified by the co-operative action of enzymatic antioxidants (e.g. superoxide dismutase, SOD; catalase, CAT; glutathione peroxidase, GPOX; glutathione-s-transferase, GST; and glutathione reductase, GR) and by non-enzymatic antioxidants such as glutathione (GSH) (Ahmad, 1995; Bergamini et al., 2004; Halliwell, 1987; Halliwell and Chirico, 1993; Hermes-Lima, 2005; Lesser, 2006). In addition to the antioxidant systems described above, the glyoxylase pathway also plays an important role during oxidative stress, by detoxifying reactive aldehydes including methylglyoxal (Kalapos, 2008; Thornalley, 1990). This pathway is comprised of two enzymes glyoxylase I (GLX-I) and glyoxylase II (GLX-II), which act together to catalyse the breakdown of the methylglyoxal formed as a byproduct of several metabolic pathways, including glycolysis, and from lipid peroxidation caused by excess ROS production (Kalapos, 2008; Thornalley, 1990). Removal of methylglyoxal is very important for the recovery of organisms following an oxidative stress event. In small estuaries, heavy rain events can reduce the salinity of the water column, a condition that can be prolonged for several
n estuary (Southern Chile) low days. For example, in the Quempille salinity conditions can occasionally remain for up to 72 h after a heavy rain event (Chaparro et al., 2008c), greatly impacting on the behaviour and physiology of some molluscan estuarine species (e.g. bivalves, Ostrea chilensis; gastropods, Crepipatella dilatata, Chaparro et al., 2009). Crepipatella dilatata is an estuarine gastropod that inhabits estuarine areas in Southern Chile. This gastropod broods its encapsulated embryos in the pallial cavity under the female shell for protection (Collin, 2003; Chaparro et al., 1998). This location provides favourable environmental conditions and protects the brooded developing embryos from osmotic changes during the estuarine low salinity events (Chaparro et al., 2008a). Although in the first instance this reproductive mode seems to be beneficial for the isolated brooded embryos, the female pallial fluid bathing the encapsulated embryos starts to change. The levels of excretory products increase in the pallial fluid, the pH level drops and the dissolved oxygen content rapidly declines (Chaparro et al., 2009), with oxygen availability eventually falling to anoxic levels after 12 h. Segura (2012), demonstrated that the magnitude of oxygen

deprivation in females and brooded embryos is related to the isolation period and also to the embryos developmental stage within the pallial cavity. Thus, the pronounced periodical oxygen changes in oxygen status of the fluid in pallial cavity of C. dilatata raises the question, what antioxidant strategy allows females (nonbrooding and brooding) and brooded embryos to live under the threat of oxidative stress? Additionally, to our knowledge, no information exists regarding the cellular implications of oxygen debt in non-brooding and brooding females, incubating embryos at different developmental stages, after prolonged periods of anoxia considering the high energetic costs that advanced embryos have as a result of their increased metabolism (Brante, 2006; Brante et al., 2008; MaedaMartínez, 2008; Segura et al., 2010) and/or intrinsic processes like intracapsular metamorphosis (Segura et al., 2010). Previous studies have indicated that most mollusc species are anoxia-tolerant due in part to an ability to up-regulate antioxidant metabolism during hypoxia to avoid increased oxidative damage during reoxygenation process (Gorr et al., 2010; Hermes-Lima et al., 1998; Pannunzio and Storey, 1998). Thus, C. dilatata might have elevated antioxidant metabolism to help cope with periods of prolonged oxygen deprivation. Additionally, important ontogenetic increases in antioxidant metabolism might also be present during embryo development, considering the increased oxygen demand during development, particularly when reaching the juvenile stage at hatching time (Segura et al., 2010). To address the above questions, we investigated the cellular mechanisms that allow females of C. dilatata (brooding and nonbrooding) and their respective embryos (early embryonic stage, veliger intermediate stage and advanced pre-hatching stage) to tolerate drastic changes in oxygen concentrations in the pallial cavity, as a consequence of female isolation due to salinity reductions in the estuary. 2. Material and methods 2.1. Experimental specimens and treatments Individuals of Crepipatella dilatata were collected in the
n estuary (41520 S; 73 460 W, Chiloe
Island, Southern Quempille Chile) during early spring 2012 (September, South hemisphere) and immediately transferred to the laboratory. Specimens were maintained in a 100 L tank with unfiltered running sea-water at a salinity of 32 PSU and a temperature of 12  C prior to carrying out the experiments. The use of unfiltered seawater allowed a continuous feeding period to the animals, considering the filter feeding capacity of this mollusc. Considering the findings of Chaparro et al. (2009), environmental salinity over 25 PSU allows in C. dilatata an active pumping of the water inside of the pallial cavity keeping a normal oxygen concentration level in it. At the beginning of the experiment (Hour 0) the salinity of the experimental aquaria was 32 PSU, salinity that allowed the females of C. dilatata to remain actively pumping seawater from the aquaria. Salinity was then gradually lowered over a period of 30 min by adding freshwater into the experimental aquaria, following the methodology used by Chaparro et al. (2009), until the salinity in the aquaria reached 10 PSU. Low salinity levels force both brooding and non-brooding females to isolate their pallial cavities from the surrounding environment. Several females of unknown reproductive status (z100 organisms) were then placed into four experimental aquaria (10 L) and were kept there for a further 72 h at low salinity to assure a state of complete hypoxia within the pallial cavity was reached (Chaparro et al., 2009). After the isolation period, the salinity of the experimental aquaria was gradually increased to 32 PSU, over a period of 30 min, by flowing unfiltered seawater into

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the aquaria. The animals were then maintained at full salinity for a further 24 h, giving a total experimental duration of 96 h from the beginning of the experiment (Fig. 1). As controls, a second group of brooding and non-brooding females were placed in four 10 L tanks with flowing seawater at constant salinity (32 PSU) for the duration of the experimental period. As detailed in Fig. 1, brooding and no-brooding females from the experimental and control aquaria were harvested throughout the experiment and tissues samples of the muscular foot of C. dilatata were collected at 0, 6, 72, 74, 84 and 96 h. Additionally, spawning masses containing embryos at early, intermediate and advanced developmental stages were collected from brooding females from the experimental and control aquaria also at 0, 6, 72, 74, 84 and 96 h (Fig. 1). Embryos were assigned to one of the following categories: (1) early embryonic stage (embryos without shell, diameter < 300 mm); (2) veliger intermediate stage (shell length 400e799 mm, with well-defined velum); and (3) advanced pre-hatching stage (close to hatching, shell length > 800 mm, foot well developed) according to Segura et al. (2010). Female foot tissue samples and embryos at different developmental stages were snap frozen using liquid nitrogen and stored at 80  C for further analysis.

2.1.1. Extraction of total protein Frozen tissue samples or embryos were ground to a fine powder in liquid nitrogen using a mortar and pestle. Total protein was extracted, on ice, by adding ice-cold enzyme extraction buffer (100 mM potassium phosphate buffer (pH 7.4), containing 0.1 mM Na2 EDTA, 1% PVPP, 1 nm Phenylmethylsulfonyl fluoride (PMSF) and 0.5% Triton-X 100) to powdered tissue at a ratio of 1:9 (w/v), followed by mixing with a vortex mixer. The homogenate was then centrifuged for 5 min at 13.000 rpm and 4  C, and the supernatant

Oxygen in the pallial

collected. The protein extracts where subjected to ultrafiltration using Sartorius Stedim Biotech (Goettingen, Germany) Vivaspin 500 ultrafiltration units (10 KD MWCO) according to the manufacturer’s instructions and semi-purified proteins were reconstituted with 100 mM potassium phosphate (pH 7.4) for the protein carbonyl and antioxidant enzyme assays, or 20 mM TriseHCl buffer (pH 7.4) for the glyoxylase assays, and were stored at 80  C prior to analysis. Protein contents were determined using the Lowry protein assay (Fryer et al., 1986) and samples were diluted, as required, with the appropriate buffer before analysis. 2.1.2. Protein carbonyl analysis Protein carbonyls levels were determined using the 2,4dinitrophenylhydrazine (DNPH) method (Reznick and Packer, 1994) adapted for use with microplates. The extinction coefficient of DNPH at 370 nm (0.022 mM1 cm1) was used to calculate the protein carbonyl content of each sample, which is expressed as nmols of carbonyls per mg protein. Assays were carried out using a Perkin Elmer Wallac Victor 1420 multilabel counter (Perkin Elmer, San Jose, California, U.S.A.), controlled by a PC, and fitted with a temperature control cell, set to 25  C, and an auto-dispenser. Data were acquired and processed using the WorkOut 2.0 software package (Perkin Elmer, San Jose, California, U.S.A.). 2.1.3. Lipid peroxide extraction and analysis Lipids were extracted by adding 300 mL of methanol:chloroform (2:1 v/v) to 50 mg fresh weight of frozen powdered tissue. The tissue was left to stand for 1 min, then 200 mL of chloroform was added and mixed for 30 s, using a vortex mixer. Deionized water (200 mL) was added and the extract was mixed again for 30 s. The resulting homogenate was centrifuged at 18,000 rpm to separate the phases. Lipid hydroperoxide levels in the extract were

32 PSU – Pallial cavity open

10 PSU – Pallial cavity isolaƟon

13 12 11 10

Back to OCRi

14

Back to Normoxia

15 Female Isolation

-1

L-1 Oxygecavity n lev(mg el inOs2id e) pallial cavity (mg O2 L )

16

32 PSU – Pallial cavity open

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9 8 7 6 5 4

Hypoxia

3 2

Anoxia

1 0 -18 -12 -6

0

6 12 18 24 30 36 42 48 54 60 66 72 78 84 90 96 102108114120 Experimental time (h)

Fig. 1. Generalized diagram showing variation of the oxygen levels in the pallial cavity of experimental females (brooding and non-brooding) at which brooded embryos are exposed (continuous line) as a consequence of salinity reduction (32e10 PSU) and salinity increase (10e32 PSU) in the experimental aquaria. Hypoxia (5 e 6 mg O2 L1) and anoxia (< 1.5 mg O2 L1) indicate oxygen levels in the pallial cavity. Striped area in the graph during the reoxygenation of the pallial cavity, indicates oxygen debt payment until reach initial oxygen consumption rates (OCRi). Horizontal dotted line indicates control females (brooding and non-brooding) and incubated embryos exposed to constant levels of salinity (32 PSU) in the experimental aquaria allowing a permanent influx of oxygen in the pallial cavity. Samples were taken in control and experimental females and their incubated embryos at 0, 6, 72, 74, 84 and 96 h. General variations in oxygen levels through time were used according to model published by Zou et al. (1996) and oxygen levels in pallial cavity of C. dilatata (Segura et al., 2016) with some modifications.

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determined using the ferric thiocyanate method (Mihaljevic et al., 1996) adapted for measurement in glass microplates and using the microtitre plate reader detailed above. A calibration curve with t-butyl hydroperoxide was used and the lipid hydroperoxide peroxide content calculated as nmol of lipid hydroperoxide/g FW. 2.1.4. Enzyme assays For the antioxidant enzymes superoxide dismutase (SOD; EC 1.15.1.1), catalase (CAT; EC 1.11.1.6), glutathione peroxidase (GPX; EC 1.11.1.9), glutathione reductase (GR; EC 1.8.1.7), glutathione Stransferase (GST; EC 2.5.1.13), glyoxylase I (GLX-I; EC 4.4.1.5) and glyoxylase II (GLX-II; EC 3.1.2.6) assay were conducted using semipurified total protein extracted as detailed above. SOD was determined via the microplate assay described by Banowetz et al. (2004) with minor modifications (Cubillos et al., 2014). A 50 mL of protein extract, diluted protein extract or standard [prepared from bovine liver SOD (SigmaeAldrich, St. Louis, MO, U.S.A.) where one unit of SOD corresponded to the amount of enzyme that inhibited the reduction of cytochrome c by 50% in a coupled system with xanthine oxidase at pH 7.8 and 25  C] was mixed with 125 mL of freshly prepared reaction solution containing piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES) buffer, pH 7.8, 0.4 mM o-dianisidine, 0.5 mM diethylenetriaminepentaacetic acid (DTPA) and 26 mM riboflavin. Absorbance at 450 nm (A450) was measured immediately (t ¼ 0 min) then samples illuminated with an 18 W fluorescent lamp placed 12 cm above the plate for 30 min and A450 measured again (t ¼ 30 min). A regression analysis was used to prepare a standard line relating SOD activity to the change in A450. Superoxide dismutase activities in the extracts, calculated with reference to the standard line, were expressed as units SOD mg1 of total protein [prepared from bovine liver SOD (SigmaeAldrich, St. Louis, MO, U.S.A.) where one unit of SOD corresponded to the amount of enzyme that inhibited the reduction of cytochrome c by 50% in a coupled system with xanthine oxidase at pH 7.8 and 25  C] was mixed with 125 mL of freshly prepared reaction solution containing piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES) buffer, pH 7.8, 0.4 mM o-dianisidine, 0.5 mM diethylenetriaminepentaacetic acid (DTPA) and 26 mM riboflavin. CAT was assayed using the chemiluminescent method of Maral et al. (1977), as adapted by Janssens et al. (2000) for 96-well microplates. 50 mL of protein extract, diluted protein extract or standard [purified bovine liver CAT (SigmaeAldrich, St. Louis, MO, U.S.A.) in homogenization buffer] was mixed with 100 mL of 100 mM phosphate buffer (pH 7.0) containing 100 mM Na2 EDTA and 50 mL of 1 mM H2O2. Samples were then incubated at 25  C for 30 min, after which 50 mL of a solution containing 20 mM luminol and 11.6 units mL1 of horseradish peroxidase (SigmaeAldrich, St. Louis, MO, U.S.A.) was injected into each well. Light emission, the intensity of which was proportional to the amount of H2O2 remaining in the mixture, was measured. A regression analysis was used to prepare a standard line relating standard CAT activities to the intensity of light emission. Catalase activities in the extracts were calculated with reference to the standard line and expressed as mM of H2O2 consumed min1 mg1 of total protein. GPOX was assayed using the spectrophotometric method of Paglia and Valentine (1967) with modifications for use with a microplate reader. GR was assayed using the method of Cribb et al. (1989). 50 mL of protein extract, diluted protein extract or a standard obtained from wheat germ in homogenization buffer (SigmaeAldrich, St. Louis, MO, U.S.A.) was mixed with 150 mL of 100 mM sodium phosphate buffer (pH 7.6) containing 0.1 mM 5,50 -dithiobis (2-nitrobenzoic acid) (DTNB) and 10 mL of NADPH (10 mg/ml; 12 mM). The reaction was initiated by the injection of 10 mL of oxidized glutathione (GSSG) (1 mg/ml; 3.25 mM) and the absorbance at 415 nm (A415)

was measured every 30 s for 3 min, with the plate shaken automatically before each reading. The rate of increase in A415 per minute was calculated and a regression analysis was used to prepare a standard line relating standard GR activities to the change in A415. GR activities in the extracts were then calculated with reference to the standard line and expressed as nmoles of oxidized glutathione reduced per min per milligram of total protein. GST was determined using the method of Habig et al. (1974), modified by Brogdon and Barber (1990) for use in a microplate reader. A 20 mL of diluted extract or standard was mixed with 170 mL of assay buffer containing 50 mM Tris-HCl buffer (pH 7.6), 5 mM Na2 EDTA, 0.14 mM NADPH, 1 mM GSH and 3 units mL1 glutathione reductase (from wheat germ, SigmaeAldrich, St. Louis, MO, U.S.A.; EC 1.6.4.2). The reaction was initiated by the addition of 20 mL t-butyl hydroperoxide to give a final concentration of 0.2 mM. The consumption of NADPH was monitored at 340 nm (A340) every 30 s for 3 min, with the plate shaken automatically before each reading. The GPOX activities in the extracts were calculated with reference to a standard line constructed with GPOX purified from bovine erythrocytes (SigmaeAldrich, St. Louis, MO, U.S.A.) in extraction buffer. Data are expressed as nmols min1 mg1 of total protein. The activity of GLX-I was determined according to Hossain et al. (2010), and activity of GLX-II was determined according to the method of Principato et al. (1987), with minor modifications. For GLX-I the assay mixture contained 100 mM potassium phosphate buffer (pH 7.0), 15 mM magnesium sulfate, 1.7 mM GSH and 3.5 mM Methylglyoxal (MG) and 25 mL of protein extract, diluted protein extract or standard (prepared from (G4252) GLX-I from Saccharomyces cerevisiae (SigmaeAldrich, St. Louis, MO, U.S.A.)), in a final volume of 200 mL. The reaction was started by the addition of MG and the increase in absorbance was recorded at 240 nm for 2 min. GLX-II activity was determined by monitoring the formation of GSH at 410 nm for 2 min. The reaction mixture contained 100 mM TriseHCl buffer (pH 7.2), 0.2 mM DTNB and 1 mM S-D-lactoylglutathione (SLG) and 25 mL of diluted protein extract, in a final volume of 200 mL. The reaction was started by the addition of SLG and the activity was calculated using the extinction coefficient of 13.6 mM1 cm1, which was corrected for path-length. Enzymes assays were carried out using a PerkinElmer (Wallac) 1420 multilabel counter (Perkin Elmer, San Jose, California, U.S.A.), as detailed above, except for Glx-I for which assays were conducted using a Jasco (Jasco, Tokyo Japan) 550 spectrophotometer fitted with temperature control. Controls included heat denatured samples and assays conducted in the absence of added substrates. 2.2. Statistical analysis When the normality and homogeneity of variance of data were verified, we used a 3-way ANOVA to identify significances of changes in oxidative damage (lipid peroxides and protein carbonyls) and antioxidant levels (SOD, CAT, GPOX, GR, GST, GLX-I and GLX-II) through the time (0, 24, 48, 72, and 96 h) in brooding (brooding early embryonic stage, veliger intermediate stage and advanced pre-hatching stage) and no-brooding females induced to an isolation of the pallial cavity caused by salinity reduction (control salinity: 32 PSU constant through the time/and treatment of salinity reduction: between 0 and 0.5 h (32 PSU/pallial cavity open), > 0.5 e 72 h (10 PSU/pallial cavity closed e isolation), 72.5 e 96 h ¼ pallial cavity open/32 PSU). When statistical differences between treatments were identified, a posteriori Tukey test were used to determine where differences of oxidative damage and antioxidant levels were significant. Statistical analyses were carried out using Statistica® V 7.0 software with a significance level p  0.05.

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3. Results 3.1. Oxidative damage and antioxidant responses in brooding and non-brooding female tissues The reproductive status of brooding and non-brooding females influenced the levels of lipid peroxides (Fig. 2) and protein carbonyl levels (Fig. 3) found in the maternal tissues. Lipid peroxide levels did not change during isolation of the pallial cavity period in brooding and non-brooding females of C. dilatata. During oxygen reduction period (hypoxia/anoxia), an increase by 2 folds in protein carbonyl levels were observed in females brooding embryos at veliger and pre-hatching stage when compared to control and nonbrooding females (Fig 3). Also, increased lipid peroxide and protein carbonyl levels were observed immediately after re-oxygenation of the pallial cavity and for the following 12 h, irrespective of reproductive condition (Figs. 2 and 3). Thus, during reoxygenation period, lipid peroxides levels increased 9, 7, and 5-folds in experimental females brooding early, veliger and pre-hatching embryos respectively, when compared to control animals. Similar results were observed for protein carbonyls levels, which increased by 3, 3, and 2 folds in experimental females brooding early, veliger and pre-hatching embryos respectively, when compared to control animals during re-oxygenation (Figs. 2 and 3). After re-oxygenation, lipid peroxidation and protein carbonyls levels in non-brooding females increased 11 and 4-folds respectively when compared to control animals (Figs. 2 and 3). It is important to highlight that both types of oxidative damage levels in brooding females were always greater

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than those observed in non-brooding females, either during isolation of the pallial cavity and/or after its re-oxygenation. Lipid peroxide and protein carbonyl levels in brooding and none-brooding females, had returned to baseline levels after 24 h following reoxygenation (Figs. 2 and 3). Female tissues generally showed higher levels of lipid peroxides (Fig. 2) and protein carbonyls (Fig. 3) than their respective incubated embryos. The reproductive condition of treated females of C. dilatata did not appear to influence levels of SOD (Fig. 4), CAT (Fig. 5), GPOX (Fig. 6), GR (Fig. 7), GST (Fig. 8), GLX-I (Fig. 9) and GLX-II (Fig. 10). However, prolonged isolation of the pallial cavity (hypoxia/anoxia) significantly decreased SOD (Fig. 4), CAT (Fig. 5), GPOX (Fig. 6), GR (Fig. 7), GST (Fig. 8), GLX-I (Fig. 9) and GLX-II (Fig. 10) activities when experimental females of C. dilatata were compared to control females. A significant increase in decreased SOD (Fig. 4), CAT (Fig. 5), GPOX (Fig. 6), GR (Fig. 7), GST (Fig. 8), GLX-I (Fig. 9) and GLX-II (Fig. 10) levels were only observed 12 h after re-oxygenation of the pallial cavity, in both brooding and non-brooding females (Fig. 4e10). 3.2. Oxidative damage and antioxidant response in encapsulated embryos Our results showed that lipid peroxides (Fig. 2) and protein carbonyls (Fig. 3) levels in embryos exposed to isolation of the pallial cavity followed by re-oxygenation, are influenced by developmental stage. During the isolation (hypoxia/anoxia) period, only brooded embryos in pre-hatching development stage showed

Fig. 2. Lipid peroxides levels in brooding and non-brooding females of C. dilatata and their respective brooded encapsulated embryos (early stage, intermediate veliger stage and pre-hatching stage) during the isolation and re-opening period of the pallial cavity. Black filled bars represent experimental organisms; Grey filled bars represent control individuals; Dotted area in the graph represent the isolation period of the pallial cavity. Error bars represent ± SD. Column with different letters have significantly different means (Tukey HSD, P < 0.05). Time 0 h indicates baseline oxygen levels, time 6 and 72 h indicates isolation period of the pallial cavity; time 74, 82 and 96 h indicates re-oxygenation period of the pallial cavity (n ¼ 48/experiments were replicated 4 times).

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Fig. 3. Protein carbonyls levels in brooding and non-brooding females of C. dilatata and their respective brooded encapsulated embryos (early stage, intermediate veliger stage and pre-hatching stage) during the isolation and re-opening period of the pallial cavity. (n ¼ 48/experiments were replicated 4 times).

Fig. 4. SOD levels in brooding and non-brooding females of C. dilatata and their respective brooded encapsulated embryos (early stage, intermediate veliger stage and pre-hatching stage) during the isolation and re-opening period of the pallial cavity. (n ¼ 48/experiments were replicated 4 times).

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Fig. 5. CAT levels in brooding and non-brooding females of C. dilatata and their respective brooded encapsulated embryos (early stage, intermediate veliger stage and pre-hatching stage) during the isolation and re-opening period of the pallial cavity. (n ¼ 48/experiments were replicated 4 times).

Fig. 6. GPOX levels in brooding and non-brooding females of C. dilatata and their respective brooded encapsulated embryos (early stage, intermediate veliger stage and prehatching stage) during the isolation and re-opening period of the pallial cavity. (n ¼ 48/experiments were replicated 4 times).

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Fig. 7. GR levels in brooding and non-brooding females of C. dilatata and their respective brooded encapsulated embryos (early stage, intermediate veliger stage and pre-hatching stage) during the isolation and re-opening period of the pallial cavity. (n ¼ 48/experiments were replicated 4 times).

Fig. 8. GST levels in brooding and non-brooding females of C. dilatata and their respective brooded encapsulated embryos (early stage, intermediate veliger stage and pre-hatching stage) during the isolation and re-opening period of the pallial cavity. (n ¼ 48/experiments were replicated 4 times).

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Fig. 9. GLX-I levels in brooding and non-brooding females of C. dilatata and their respective brooded encapsulated embryos (early stage, intermediate veliger stage and prehatching stage) during the isolation and re-opening period of the pallial cavity. (n ¼ 48/experiments were replicated 4 times).

Fig. 10. GLX-II levels in brooding and non-brooding females of C. dilatata and their respective brooded encapsulated embryos (early stage, intermediate veliger stage and prehatching stage) during the isolation and re-opening period of the pallial cavity. (n ¼ 48/experiments were replicated 4 times).

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significant increases in lipid peroxide and protein carbonyl levels by 3 and 2-folds respectively, when compared to controls. In contrast, during re-oxygenation of the pallial cavity, early, veliger and pre-hatching developmental stage embryos showed a 7, 5 and 8-fold increases in lipid peroxides respectively (Fig. 2). Similarly, during the same experimental period, protein carbonyls increased 2, 2 and 3-folds in early, veliger and pre-hatching developmental stage embryos. (Fig. 3). Oxidative damage levels in brooded embryos (all developmental stages), remained high during the next 24 h post-re-oxygenation, by which time they had declined to control values. Significant differences in the activities of SOD, CAT, GPOX, GR, GST, GLX-I and GLX-II were observed when treated and control embryos were compared over time (Figs. 4 e 10). During the period that the pallial cavity was isolated, embryos irrespective of their developmental stage, showed a significant reduction in antioxidant enzyme activities. Although antioxidant defences did not responds immediately followed re-oxygenation, most embryos showed increases in SOD (Fig. 4), CAT (Fig. 5), GPOX (Fig. 6), GR (Fig. 7), GST (Fig. 8), GLX-I (Fig. 9) and GLX-II (Fig. 10) after a period of time from 12 to 36 h post re-oxygenation. 4. Discussion Our results indicate that brooding, as the reproductive mode of C. dilatata, causes oxidative damage in females and incubated embryos following oxygen limitation. Females brooding embryos in veliger and pre-hatching stages, exposed to oxygen deprivation, show considerable oxidative damage after environmental isolation, compared to non-brooding females. As a consequence of maternal isolation, caused by low salinity, the availability of oxygen in the pallial cavity is reduced, resulting in oxygen becoming a limited resource. It is well known that brooding conditions can reduce oxygen levels severely, eventually resulting in hypoxic conditions. For example, adults of Ostrea chilensis and C. dilatata brooding embryos can reduce oxygen levels in the pallial cavity until hypoxic levels (< 1.5 mg O2 L1) in 0.5 and 12 h respectively since the isolation of the pallial cavity triggered by salinity reduction (Chaparro et al., 2009). It had been demonstrated that hypoxia causes intertidal molluscs to slow their heart rates when exposed at low tides (Trueman, 1967). As the heart rate of molluscs is directly associated with oxygen uptake (Marshall and McQuaid, 1992), metabolic depression can limit the amount of oxygen needed by tissues, and this can help to limit ROS formation (Hermes-Lima et al., 1998). This appears to be the case in the present study, as during isolation of the pallial cavity in C. dilatata, lipid peroxide levels remained similar to control levels in both maternal tissues and brooded embryos. These findings support the general hypothesis that a reduction in metabolism under conditions of oxygen limitation can limit ROS production and therefore oxidative damage (Davies, 2000). However, it is interesting to note that while lipid peroxide levels did not increase during the isolation period and protein carbonyls only showed a small increase in intermediate veliger and prehatching stage embryos. This could have been due to reduced protein turnover rather that increased oxidative damage. It has been shown that during low oxygen induced metabolic depression, invertebrates can switch to anaerobic metabolism to sustain basic cellular functions (Akberali and Trueman, 1985; Storey, 1993) and that this enables continued ATP production at levels sufficient to maintain cell viability (de Zwaan and Wijsman, 1976; Storey, 1993). It has been shown that tissue of the female foot (brooding and non-brooding) and brooded embryos of the estuarine Chilean oyster Ostrea chilensis can switch to anaerobic metabolism soon after isolation of the pallial cavity, indicated by the accumulation of

lactate (Segura et al., 2015). It has also been shown in O. chilensis that prolonged periods of isolation from the outside environment can result in acidification of the fluid contained within the pallial cavity, probably due to an increase in CO2 levels (Chaparro et al., 2009). Although the steady state level of CO2 in the pallial cavity can generate unfavourable conditions for both females and incubated embryos of C. dilatata, a lower pH may also help to limit ROS generation. For example, Styf et al. (2013) showed that reducing pH level in the water culture of early developmental stages of embryos of the crustacean Nephrops norvegicus reduce significantly oxidative damage (protein carbonyl levels) when compared to control organisms. Similar results were observed when haemocytes of the oyster Crassostrea virginica were exposed to an acid pH and low oxygen levels, a 50% reduction in ROS production was observed (Boyd and Burnett, 1999). Reduced ROS production during anoxia has also been shown to be associated with levels of the protein ferritin, which is an intracellular protein that stores and controls the release of iron. Ferritin can help to limit HO
generation caused by Fenton reactions that occur in the presence of transition metals such as Fe3þ (Larade and Storey, 2004). Interestingly, although levels of oxidative damage remained relatively low in both female tissues and embryos during pallial cavity isolation, no significant increases in the activities of enzymes associated with antioxidant metabolism (SOD, CAT, GLX-I, GLX-II, GPOX, GR and GST) were observed during the period of isolation. In fact small reductions in activities were observed for some enzymes. A similar trend was observed for the intertidal periwinkle Littorina littorae, which showed reduced SOD, CAT, GPOX, GR and GST, activities of 44% e 70%, when animals were exposed to anoxic conditions for a 6 day period (Pannunzio and Storey, 1998). Similar result were found for the mussel Mytilus galloprovincialis, which when exposed to hypoxic conditions for more than 72 h showed significant reductions in CAT and GST gene expression (Woo et al., 2013). In this context, it is important to highlight that ROS participate as signalling molecules associated to antioxidant upregulation mechanisms (Hermes-Lima, 2004; Lesser, 2006) It has been observed that ROS can via the activation of transcription factors regulate genes, resulting increased levels of antioxidant compounds (Hensley et al., 2000). When environmental salinity is rapidly increased to 32 PSU, after 72 h of environmental isolation, the influx of water rich in dissolved oxygen into the pallial cavity resulted in greatly increased levels of oxidative damage in both adults and encapsulated embryos. This was most likely due a large increase in ROS levels due to the rapid resumption of oxidative metabolism coupled to the loss of antioxidant capacity that occurred during the period of low oxygen (Hermes-Lima et al., 1998). It is likely that the longer the period of insulation (low oxygen) to which C. dilatata is exposed, the greater the physiological challenge caused by oxidative damage (Burnett and Stickle, 2001; Halliwell and Gutteridge, 2007), due to a possible oxygen debt. For example, Segura et al. (2016) determined that prolonged isolation periods (48 h) in brooding females of C. dilatata generate oxygen debts of 4.5 mg O2 g1 once the pallial cavity is newly open to the surrounding environment, whereas, oxygen debt in brooded embryos depends on the development stage in a dependant manner, varying between 0.004 and 0.024 mg O2 g1. The increase in oxidative damage in brooded encapsulated embryos of C. dilatata at later developmental stages is most likely associated with increases in their oxygen requirements. A previous study showed that the oxygen consumption rate (OCR) in advanced and pre-hatching embryos of C. dilatata increased by 154% and 114% respectively, when compared to early embryos and veliger developmental stages (Segura et al., 2010). As development proceeds the internal structure of the capsule changes with a reduction in the

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spongy layer, probably due to increased embryo activity, and this results in increased permeability to oxygen and hence more oxygen in the intracapsular fluid (Segura et al., 2010). Hence, the more developed the embryos inside the capsules are, the thinner the walls of the capsules become (Brante et al., 2008; Ojeda and Chaparro, 2004; Segura et al., 2010), a situation that could favour more rapid diffusion of oxygen into the intracapsular fluid. In addition, differences in the biochemical composition of embryos during development might also influence their tolerance to ROS during re-oxygenation of the pallial cavity. A recent investigation has shown that there is a 3-fold increase in ROS levels in brooded embryos of the sea star Anasterias antarctica as development progresses and that this is due to a reduction in liposoluble antioxi
rez et al., 2015). dants such as a-tocopherol and b-carotene (Pe The lack of a physical barrier (capsule) in adults of C. dilatata allows females to remain in direct contact with the oxygenated water that enters into the pallial cavity, once environmental conditions become favourable, this allows a more rapid switch back to aerobic metabolism. Consequently, this situation could result in a considerable physiological cost to the organism, considering the oxygen debt generated during a prolonged isolation period of the pallial cavity. For example, it was observed that prolonged periods of anoxia can generate considerable oxygen debts in the gold fish Carassius auratus, causing an increase in the OCR from 15 to 33.5 mg O2 * 100 g1 (Thillart and Verbeek, 1991). It is also well known, that increases in the OCR can cause the accumulation of ROS in cells and that this result in oxidative damage (Abele et al., 2012). It is possible that the elevated levels of oxidative damage found in females and incubated embryos of C. dilatata after re-oxygenation of the pallial cavity could be a consequence of what is in effect an oxygen debt payment, but this requires further investigation. A recent investigation has shown that the mussel M. edulis when exposed to anoxia for 72 h followed by a 24 h re-oxygenation period, can generate a 2fold increase in superoxide anion levels, during the payment of the oxygen debt (Rivera-Ingraham et al., 2013). Significant increases in lipid peroxidation, from 35 to 52 nmol g1 in the muscle and from 45 to 70 nmol g1 in the gill tissue have also been observed in the white shrimp Litopeneaus vannamei after transfer from 24-h under hypoxic conditions to normoxia (Zenteno-Savín et al., 2006). As a response to oxidative stress after re-oxygenation of the pallial cavity, females and incubated embryos of C. dilatata rapidly up-regulate their antioxidant metabolism, seen as increases in SOD, CAT, GPOX and GR activities. Similar results were observed in clam (Chamelea gallina) haemocytes, which after being exposed to anoxia for 48 h showed significant increases in total SOD levels, from 50 to 79 U mg protein1, 24 h after re-oxygenation (Monari et al., 2005). Although the enzymes associated with antioxidant metabolism analysed in C. dilatata did not respond within 2 h of reoxygenation, levels rapidly increased in a synchronous manner within 24 h of re-oxygenation of the pallial cavity and levels of oxidative damage returned to control levels during this period. In addition to measuring the levels of enzymes directly or indirectly involved in ROS scavenging (SOD, CAT, GPOX, GST and GR), we also measured the activities of GLX-I and GLX-II. The importance of these enzymes as part of the metabolic processes that cells use to overcome an oxidative stress event is often overlooked. To our knowledge, there have been not detailed studies investigating the activity of the glyoxalase pathway in brooding gastropods exposed to oxygen deprivation followed by reoxygenation. Methylglyoxal is not only highly cytotoxic, but previous studies have shown that failure to remove methylglyoxal can promote H2O2 formation, an effect that is dose-dependent (Leoncini and Poggi, 1996; Thornalley, 1993). Twelve hours after re-oxygenation of the pallial cavity the activities of both GLX-I and GLX-II increased significantly in both

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females and incubated embryos, with a decline in oxidative damage also observed. It is likely that the increased activities of these two enzymes would eliminate any methylglyoxal induced oxidative stress occurring due to re-oxygenation. Both the cytotoxic effects of methylglyoxal and any potential increases in H2O2 formation due to the presence of methylglyoxal would be eliminated and so less ROS would need to be neutralised by the antioxidant systems present. In C. dilatata, both the glyoxylases and the enzymes associated with antioxidant metabolism did not immediately increase following reoxygenation, with no significant increases observed 2 h after pallial cavity opening. Micro calorimetric studies carried out in the estuarine oyster Crassostrea virginica showed a reduction in energy consumption from 3.16 J g dry wt1 h1 to 2.38 J g dry wt1 h1 while animals were transferred from normoxia to an anoxic environment (Stickle et al., 1989). A severe reduction in energy consumption of over 90% (from 8.76 to 0.78 J g dry wt1 h1) was also observed in the gastropod Thais haemastoma under the same physiological conditions (Stickle et al., 1989). Therefore, the delay in the response to elevated oxidative damage levels in adults and embryos of C. dilatata may have been due an energetic requirement to increase glyoxylase and antioxidant defences levels.

5. Conclusions We conclude that, repeated reductions in salinity in estuarine systems caused by prolonged periods of rain can have consequences for the physiology of females and incubated embryos of C. dilatata. During pallial isolation, caused by reduced salinity levels, there is a decline in available oxygen levels pallial cavity of females and in incubated embryos that forces them to depress their metabolism and shift from aerobic to anaerobic metabolism, which could potential help to limit ROS generation. Following reoxygenation, a shift back to aerobic metabolism occurs and both glyoxalase pathway and general antioxidant metabolism are upregulated in response to ROS production elevated levels of oxidative damage to lipids and proteins. This increase in antioxidant defences allows females, irrespective of their reproductive condition, and incubated embryos, at an advanced developmental stage, limit further oxidative damage and over the next 12 e 24 h the levels of lipid peroxides and protein carbonyls fall to basal levels. Embryos at the early and veliger stages of development can even more effectively cope with oxidative stress and with oxidative damage returning to basal levels less than 12 h post reoxygenation. It is important to note that in C. dilatata, antioxidant defence levels and glyoxylase metabolism both increase rapidly, within 12 h, following reoxygenation of the pallial cavity and that defensive metabolism is maintained for at least the next 24 h. From this study, it appears that C. dilatata is a species capable of rapidly changing several metabolic pathways, which enables it to tolerate the repeated oxidative stress events that occur as a consequence of frequent variations in the levels of oxygen found in the pallial cavity, brought about by changes in salinity as consequence of heavy rain during winter, or strong tidal changes in estuarine environments. The present study was carried out over extended time intervals and so as to assess the physiological response to hypoxia/anoxia (during the isolation period) and then re-oxygenation. Future studies over shorter time-scales will be important to further understand the importance of antioxidant metabolism in females and encapsulated embryos of C. dilatata, particularly under field conditions. Additionally, the levels of ROS and non-enzymatic antioxidants should also be measured during periodical changes in oxygen levels in the pallial cavity of C. dilatata.

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Acknowledgement All experiments were carried out at Calfuco Marine Laboratory e Universidad Austral de Chile, while oxidative damage and antioxidant analyses were carried out at the Department of Botany e University of Otago. We thanks all at these laboratories that assisted in this work. ORC thanks FONDECYT (Fondo Nacional de Inves
n Científica y Tecnolo
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Abbreviations COX: cytochrome oxidase ROS: reactive oxygen species SOD: superoxide dismutase CAT: catalase GPOX: glutathione peroxidase GST: glutathione-s-transferase GR: glutathione reductase GSH: glutathione IWC: internal wall capsule OCR: oxygen consumption rate