Photochemistry and Photobiology, 2014, 90: 1314–1323

Cellular Changes Associated with the Acclimation of the Intertidal Sea Anemone Actinia tenebrosa to Ultraviolet Radiation Victor M. Cubillos*1,2,3§, Miles D. Lamare2, Barrie M. Peake3 and David J. Burritt1 1

Department of Botany, University of Otago, Dunedin, New Zealand Department of Marine Science, University of Otago, Dunedin, New Zealand 3 Department of Chemistry, University of Otago, Dunedin, New Zealand 2

Received 20 March 2014, accepted 16 June 2014, DOI: 10.1111/php.12310

ABSTRACT To assess the relative importance of long- and short-term cellular defense mechanisms in seasonally UV-R-acclimated Actinia tenebrosa (Anthozoa, Actiniidae), individuals were exposed to summer doses of PAR, UV-A, UV-B and enhanced UV-B (20%) for a period of 4 days. Mycosporinelike amino acids (MAAs) and cyclobutane pyrimidine dimer (CPD) concentrations were quantified, while oxidative damage to lipids and proteins, and the activities or levels of the antioxidant enzymes SOD, CAT, GR, GPOX and total glutathione were determined. Our results show that summer UV-R-acclimated individuals had a higher UV-R tolerance, with no significant increases in CPDs levels, than winteracclimated sea anemones possibly due to higher MAA concentrations. Summer-acclimated individuals showed increased lipid and protein oxidation and GPOX activity only when they were exposed to UV-B at 20% above ambient UV-R levels. In contrast, winter-acclimated sea anemones showed elevated levels of oxidative damage, GPOX and SOD activities after exposure to UV-A or UV-B at ambient and elevated levels. Thus, this study indicates that long-term UV-R acclimation mechanisms such as the accumulation of MAAs could be more important than short-term increases in antioxidant defenses with respect to reducing indirect UV-R damage in intertidal sea anemones.

INTRODUCTION Over the past 50 years, stratospheric ozone concentrations have declined significantly in polar and mid-latitude regions (1–3), as a consequence of the use of chlorine and bromide containing substances. While modeling of these changes in the ozone layer have projected a partial recovery of the ozone layer over the next 2–3 decades, due to the transport of ozone-poor air from Antarctica (4), large differences in the UV-B (290–320 nm) radiation doses have been observed at mid-latitudes when the Southern and Northern hemispheres are compared (5). For example, Lauder (46°S, New Zealand) receives 50% more UV-B radiation than Garmisch (47°N, Germany) during the summer season (5). *Corresponding author email: [email protected] (Victor M. Cubillos) § Current address: Instituto de Ciencias Marinas y Limnol
ogicas, Universidad Austral de Chile, Valdivia, Chile © 2014 The American Society of Photobiology

Furthermore, small-scale ozone depletion events known as mini ozone holes can reduce the total ozone column by 40%, increasing the penetration of UV-B at mid-latitudes (6–8). Hence, elevated levels of UV-B are a continuing problem for marine organisms living in parts of the Southern hemisphere. While ozone depletion does not change incident PAR (400– 750 nm) and UV-A (320–400 nm), it enhances the levels of UV-B radiation reaching the Earth’s surface and can result in photo-chemical and photo-biological effects on marine organisms (9). UV-R can cause damage to DNA, proteins and lipids both directly and indirectly (10). Direct UV-B absorption causes structural damage to DNA mainly through the formation of pyrimidine dimers (cyclobutane pyrimidine dimers [CPDs] or 6-4 photoproducts [6-4PP]), which if not repaired can influence the growth, development and reproduction of marine organisms (11–14). In general, 90% of the pyrimidine dimers formed are CPDs with only 10% 6-4PP (15), with the level of CPDs being largely UV-B dose dependent (16). In addition, UV-R can damage marine organisms indirectly through the generation of reactive oxygen species (ROS). Normally ROS are generated at low levels as by-products of aerobic metabolism in living organisms; however, their production can be greatly increased due to exposure to stressors including UV-R, as has been demonstrated in marine organisms such as cyanobacteria (17), phytoplankton (18), macroalgae (19–22) and invertebrates (23–25). UV-R can generate ROS by energetic transfer between a photosensitized molecule and ground state oxygen, a situation that leads to the generation of single oxygen (1O2) and the subsequent generation of the superoxide radical (O
2 ), hydrogen peroxide (H2O2) and the highly damaging hydroxyl radical (OH
) (26). ROS can subsequently cause damage to the key cellular macromolecules, lipids, proteins and DNA when they accumulate to levels that exceed the capacity of a cell to remove them, a condition commonly known as oxidative stress (27). It has been shown experimentally that intertidal organisms can have reduced survival rates, compared with subtidal organisms, as a consequence of oxidative and DNA damage caused by exposure to elevated UV-R (28). To avoid damage by direct and indirect UV-R absorption, marine organisms have a range of defense mechanisms. Direct damage can be reduced by the presence of UV-R screening molecules, such as mycosporine-like amino-acids (MAAs), which are used by some algae, invertebrates and aquatic vertebrates to block UV-R (29–31). MAAs, which absorb UV-R between 310 and 360 nm, are synthesized

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Photochemistry and Photobiology, 2014, 90 through the Shikimate pathway by primary producers and are transferred to higher trophic levels either by direct consumption or through symbiotic relationships (32–35). Elevated levels of MAAs in marine species have been documented in organisms living in both intertidal and subtidal environments, with concentrations decreasing with increasing water depth as UV-R is attenuated (36). It has also been shown that MAAs can be transferred from adults to gametes, increasing UV-R protection during the free-living stages in the plankton (37,38). Indirect damage to DNA, lipids and proteins, caused by ROS, can be reduced in animals by increasing the levels of enzymatic anti-oxidants (e.g. superoxide dismutase [SOD], catalase [CAT], glutathione peroxidase [GPOX] and glutathione reductase [Gr]) and non-enzymatic antioxidants (e.g. glutathione [GSH]) (26). Although some cellular damage is implicit during UV-R exposure, previous investigations on marine organisms have indicated that many can increase their tolerance to UV-R through acclimation processes (39,40). Both UV-R acclimation and photoacclimation have been categorized into short- and long-term responses (41). For example, as a mechanism to reduce cellular damage under high UV-R conditions, the Antarctic microalgae (Thalassiosira sp. and Corethron criophilum) photoacclimate by increasing their levels of UV absorbing compounds (MAAs), without changing their photosynthetic rates, after only 8–12 days UV-R exposure (42). Additionally, increases in antioxidant enzymes (e.g. SOD) have been observed in the green algae Chaetomorpha linum as part of a short-term photoacclimation response to elevated UV-R levels (21). Long-term acclimation has been observed in the Antarctic krill Euphausia superba, with seasonal differences in their pigmentation levels observed. Summer-acclimated organisms showed five-fold higher UV-R screening molecule levels, than winter-acclimated organisms (43). In addition, the symbiotic sea anemone Anemonia viridis shows increased chlorophyll a levels in their zooxanthellae in response to low light levels as a photoadaptative strategy (44). While cnidarians have been used as bio-indicators of temporal variation in UV-R levels in the intertidal zone, most studies have been carried out on corals (45–47), symbiotic sea anemones (23) and bleached symbiotic sea anemones (48). Little is known about the strategies that nonsymbiotic intertidal sea anemones use to reduce cellular damage caused by exposure to UV-B and their capacity to acclimate to new environmental conditions. The few studies that have been conducted have demonstrated that nonsymbiotic sea anemones are highly susceptible to environmental stress (49), especially elevated UV-B levels (50,51). The nonsymbiotic sea anemone Actinia tenebrosa Faquhar (1898) is distributed throughout the New Zealand coastline and off shore islands, inhabiting rocky shores, and is most often found in crevices (50–52). In summer A. tenebrosa, can be exposed to very high UV-R levels when low tides are coincident with maximal UV-R levels at midday. The defense strategies that A. tenebrosa uses to reduce cellular damage and survive under these highly variable conditions are not well understood. In this study, we address the question, are mechanisms associated with long-term UV-R acclimation, such as increased levels of UV-R absorbing compounds, more import than short-term changes in cellular metabolism, such as increased antioxidant metabolism, for the survival of A. tenebrosa in a rapidly changing UV-R environment? To answer this question, biological markers of UV-R-induced direct and indirect cellular damage were compared to UV-R absorbing compound levels and

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antioxidant levels, in both late-summer and winter-collected animals, exposed to artificial UV-R under controlled environmental conditions. To assess the potential effects of ozone depletion on Actinia physiology, we also examine the same suite of biological markers in individuals exposed to environmental relevant enhanced UV-B irradiances equivalent to a 20% reduction in column ozone concentrations.

MATERIALS AND METHODS Field sampling and UV-R treatments. Forty specimens of A. tenebrosa, with basal disks ranging between 4 and 5 cm in diameter, were collected during low tide periods in the Otago Harbor, New Zealand (S 45°470 52.400 ; E 170°420 54.000 ) during late summer (March 2010) and winter (July 2010). Samples were transferred to the laboratory and any epiphytes removed. Animals were placed into 2-L PVC containers, 8 in each, containing 1 L of filtered seawater. The seawater was continuously aerated for the duration of the experiment and was changed every 2 days. The containers were randomly distributed between two controlled environment chambers (Contherm 620), set to 10°C for the winter experiment and 15°C for the summer experiment. At 12-h photoperiod every 24 h was used with the following radiation supplied: PAR (400– 700 nm) Phillips, Aquarella fluorescent tubes (250–300 lmol m
2 s
1), UV-A Phillips TL40 W fluorescent tubes (237 kJ m
2 day
1) and UVB was Phillips TL20 W fluorescent tubes (35.36and 42.12 kJ m
2 day
1 for UV-B enhanced by 20%). These levels were based upon those reported during summer time on the Otago Peninsula (53) and were determined using a portable spectroradiometer (Optronics Laboratories, Model OL754). The PAR only treatment (wavelengths >400 nm; referred to as P) was achieved by blocking wavelengths below 400 nm using a 226-UV filter film (Chris James & Co. Ltd., London, UK). The PAR+UV-A treatment (wavelengths >320 nm; referred to as PA) was achieved by blocking wavelengths below 320 nm using a 0.13 mm Mylarâ-D filter (Dupont, Dover, DE). The PAR +UV-A + UV-B treatment (wavelengths >290 nm; referred to as PAB) was achieved by blocking wavelengths below 290 nm using 0.13 mm cellulose diacetate (CDA; Lonza-Flien, Weil am Rhein, Germany). The transmission profiles of the filters used are shown in Figure S1. For the enhanced UV-B treatment (PAB-20%), which simulated a 20% ozone depletion event, the additional UV-B dose was obtained by moving the containers closer to the UV-B lamps. With this treatment, the UV-A dose was increased from 237 to 252 kJ m
2 day
1. All UV-B doses were applied for 4 h over the middle of the photoperiod. All the filters were preburned by exposing them to a full radiation spectrum for 10 h. Each radiation treatment was independently replicated three times. Actinia tenebrosa individuals were randomly sampled daily from each radiation treatment, 4 h after UV-B exposure, and were immediately wrapped in aluminum foil, snap frozen in liquid nitrogen, and stored at
80°C until required for analysis. MAAs extraction and quantification. Tissue samples were dried for 24 h in a vacuum concentrator (Speed Vac Savant—Thermo Electron Corporation), set to 45°C, and then ground to a fine powder using a bead beater (BioSpec Corporation). MAAs were extracted in 100% methanol (1 mL to 10 mg of ground tissue), mixed and the homogenate was sonicated for 8 min, using an ultrasonic water bath, and then stored, in the dark, at +4°C for 24 h. MAAs were separated and identified by HPLC/ MS using a Dionex—Ultimate 3000 HPLC/MS system (Thermo Fisher Scientific, MA) using appropriate reference standards. Prior to analysis, samples were filtered through nylon syringe filters (0.22 lm) and 20 lL of the filtered extract was injected onto a C8 Phenosphere analytical column (Phenomenex; 250 9 4.6 mm; pore size 5 lm), and eluted using a mobile phase composed of 89.9% MilliQ water + 10% MeOH + 0.1 formic acid, at a flow rate of 1 mL min
1 for 10 min. Extracts were analyzed at 310, 331, 334 and 360 nm using a UV/VIS detector and the identity of each peak was confirmed by mass spectrometry (Bruker Daltonics—TOF, MA). MAA concentrations were determined from standard curves constructed using MAA standards (>95% purity) (54). DNA extraction and CPDs quantification. DNA was extracted using a DNA Biolineâ (Bioline, MA) kit for animal tissue, according to the manufacturer’s instructions. CPDs were quantified by ELISA (enzyme-linked

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Victor M. Cubillos et al. and light treatment were the fixed factors. A Tukey’s post hoc test was carried out using Minitab V.16 with a significance level P ≤ 0.05. Log10 transformation was used as required.

immunoabsorbent assay-based system) using the method of Mori et al. (55), with minor modifications. Absorbance was measured at 405 nm (A405) using a multilabel counter (Wallac 1420, Perkin Elmer, Finland), and all reagents were obtained from Sigma-Aldrich. A calf thymus DNA standard irradiated with a known dose of UV-B radiation was made and used as a reference. Reference samples and diluted reference samples were used to assess the color development of each plate and to ensure linearity. The data presented were standardized relative to the standard calf thymus DNA absorbance value. For each DNA extract, assays was performed in triplicate and the average value was taken as representative of the sampled DNA. Measurements of oxidative damage. Tissue samples were ground to a fine powder, in liquid nitrogen, using mortar and pestle. Total protein was extracted, on ice, by homogenizing powdered tissue in extraction buffer (100 mM potassium phosphate buffer [pH 7.0], containing 0.1 mM Na2 EDTA, 1% PVPP, 1 nm PMSF and 0.5% Triton-X 100) at a ratio of 1:9 (w/v). The homogenate was centrifuged for 5 min at 27 641 g and 4°C and the supernatant was stored at
80°C prior to analysis. Protein carbonyl contents were determined in protein extracts, dialysed (10 kD MWCO) exhaustively against 100 mM potassium phosphate (pH 7.0) to remove small molecules that could interfere with the assay, according to the 2,4-dinitrophenylhydrazine method of Reznick et al. (56) adapted for microplates (25). The protein content of the extracts was determined using the Lowry protein assay (57) and the protein carbonyl content is expressed as nmol carbonyls/mg protein. Lipids were extracted by adding 300 lL of methanol:chloroform (2:1 v/v) to 50 mg FW of powdered tissue. The tissue was left to stand for 1 min, then 200 lL of chloroform was added and the suspension was homogenized, using a vortex mixer, for 30 s. Deionized water (200 lL) was added and the homogenate was mixed for 30 s and then centrifuged at 36 407 g, to separate the phases. Lipid hydroperoxide levels in the extract were determined using the ferric thiocyanate method Mihaljevic et al. (58) adapted for measurement in a microtitre plate reader. Assays were conducted using a Lil420 multilabel counter (Perkin Elmer, San Jose, CA), controlled by a PC, and fitted with a temperature control cell and an autodispenser. Data were acquired and processed using the WorkOut 2.0 software package (Perkin Elmer). Antioxidant assays. For the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and glutathione peroxidase (GPOX), total protein was extracted as detailed above. SOD was determined via the microplate assay described by Banowetz et al. (59) with minor modifications (25). CAT was assayed using the chemiluminescent method of Maral (60), as adapted by Janssens et al. (61), for 96-well microplates. GR was assayed using the method of Cribb et al. (62) with minor modifications. GPOX was assayed using the spectrophotometric method of Paglia and Valentine (63) with modifications for use with a microplate reader. Glutathione was extracted by mixing 500 lL of 5% sulfosalicylic acid with 100 mg FW of frozen powdered tissue and then homogenizing the mixture with a vortex mixer. The homogenate was then centrifuged at 18 000 rpm for 15 min at 4°C. Glutathione and glutathione disulfide levels were measured using the enzymatic recycling method, using the microtitre plate-based assay described by Rahman et al. (64). All assays were carried out using a PerkinElmer (Wallac) 1420 multilabel counter (Perkin Elmer), as detailed above. Statistical analysis. Data were analyzed to test normality and homogeneity of variance using a Shapiro–Wilk test and Levene median test, respectively. A factorial MANOVA was performed where season, dose

RESULTS MAA and CPD levels in UV-R-exposed individuals Significant differences in total MAA levels at the time of collection were found between A. tenebrosa collected during summer and winter (F1,21 = 10.37, P = 0.003), with mean levels of 8.77  0.97 and 5.23  0.39 nmol mg DW
1, respectively. HPLC/MS analysis indicated the presence of shinorine (MW + H+ = 303; kmax = 334 nm), mycosporine 2-glycine (MW + H+ = 303; kmax = 310 nm) and porphyra-334 (MW + H+ = 303; kmax = 334 nm) in A. tenebrosa in both seasons (Figure S2). Individuals sampled in summer had significantly greater levels of shinorine (P < 0.05) and mycosporine-2-glycine (P < 0.05) than individuals sampled in winter (Table 1). Levels of porphyra-334 showed no seasonal differences (Table 1). While some initial differences in MAA levels between treatment groups were observed, no significant changes in MAA levels were observed in summer or winter-acclimated individuals over the 4-day duration of the experiment (Fig. 1A,B). Individuals sampled in summer, when exposed to UV-B in the growth chambers had no significant increases in basal CPD levels at the end of the photoperiod (four hours after exposure to UV-B). In contrast, individuals sampled in winter, showed UV-B dose-dependent increases in basal CPD levels (F4,32 = 4.84, P = 0.007; Fig. 2). UV-A did not significantly increase CPD levels in either summer or winter individuals when exposed to UV-R (Fig. 2). Oxidative damage and antioxidant defenses in UV-R exposed individuals Significant differences in lipid peroxides (F12,152 = 3.67, P < 0.05) and protein carbonyls levels (F12,152 = 3.645, P < 0.05) were observed between individuals sampled in summer and winter exposed to UV-R (Fig. 3). Individuals sampled in summer and exposed to UV-A showed no significant increases in protein carbonyl and lipid peroxide levels after 4 days, compared to PAR only controls. Individuals sampled in summer and exposed to ambient and enhanced levels of UV-B at ambient levels showed significant increases in protein carbonyl (F12,152 = 3.645, P < 0.001) and lipid peroxides

Table 1. Maximum and minimum levels of shinorine, mycosporine-2 glycine and porphyra-334 in seasonal UV-R-acclimated Actinia tenebrosa at day 0 and day 4. Values of maximum and minimum MAA level are expressed in nmol mg DW
1. The proportional distribution of each MAA is also expressed as a % of total MAAs. Shinorine Summer

Mycosporine 2-glycine Winter

Summer

Porphyra-334

Winter

Summer

Winter

MAAs

Day 0

Day 4

Day 0

Day 4

Day 0

Day 4

Day 0

Day 4

Day 0

Day 4

Day 0

Day 4

Maximum Minimum % of total

12.18 1.94 83.7

11.36 1.28 85.6

7.64 1.50 89.2

7.56 1.16 90.3

2.57 0.11 7.5

1.48 0.05 7.7

0.23 0.01 1.2

0.31 0.04 1.3

1.64 0.14 8.8

1.51 0.07 6.7

0.97 0.17 9.6

0.88 0.11 8.4

Photochemistry and Photobiology, 2014, 90

Figure 1. Average (SE) of total MAA concentration nmol mg DW
1 in summer (A) and winter (B) Actinia tenebrosa exposed to P, PA, PAB and PAB-20% radiation treatments at day 0 and after 4 days exposure. Adjacent plots represent mean values of MAA concentration at the beginning (day 0) and finish (day 4) of the exposure experiment. Treatment groups with common letters are not significantly different from one another using a multiple comparison test (Tukey’s HSD) at a significance level of 0.05.

levels after 4 days (Fig. 3). In contrast, UV-A, UV-B and enhanced UV-B treatments significantly increased protein carbonyl (F12,152 = 3.645, P < 0.001) and lipid peroxide levels (F12,152 = 3.67, P < 0.05) in individuals sampled in winter, compared to PAR only controls (Fig. 3). Individuals sampled in summer showed significantly higher initial GPOX (t22 = 9.682), total glutathione (t22 = 7.467) and GSH (t22 = 5.645) activities than those sampled in winter. UVR treatments had no significant influences on SOD, CAT and GR activities in individuals sampled in summer, compared to PAR only controls (Fig. 4). GPOX activities in individuals sampled in summer and exposed to UV-B were significantly higher after 4 days, compared to individuals not exposed to UV-B (F12,152 = 2.056, P < 0.05; Fig. 4). UV-R treatments caused a significant increase in GPOX activities in individuals sampled in winter, compared to PAR only controls, with UV-B causing a greater increase than UV-A (F12,152 = 2.056, P < 0.05; Fig. 4). UV-R treatments had no significant influence on CAT and GR activities in individuals sampled in winter (Fig. 4). Both total glutathione and GSH levels were significantly influenced by UV-R. Individuals sampled in summer and exposed to UV-B showed significant increases in both total glutathione (F12,152 = 15.70, P < 0.05) and GSH (F12,152 = 2.29, P < 0.05) levels while UV-A had no influence. In contrast, individuals sampled in winter showed significant increases in both total

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Figure 2. Average (SE) CPD absorbance in summer (A) and winter (B) UV-B-collected Actinia tenebrosa exposed to four radiation treatments (P [PAR], PA [PAR + UVA], PAB [PAR + UVA + UVB] and PAB-20% [PAR + UVA + UVB-20%]). Concentrations are measured at the beginning of the experiment and after 4 days. Treatment groups with common letters are not significantly different from one another using a multiple comparison test (Tukey’s HSD) at a significance level of 0.05.

glutathione (F12,152 = 15.70, P < 0.05) and GSH (F12,152 = 2.29, P < 0.05) levels in response to both UV-A and UV-B (Fig. 5). In individuals sampled in summer, a significant transient increase in GSSG (F12,152 = 4.97, P < 0.05) was observed for the elevated UV-B treatment, but GSSG levels returned to the initial levels by the end of the experiment. In contrast, exposure to UV-B caused a sustained increase in GSSG levels in individuals sampled in winter (F12,152 = 4.97, P < 0.05), while UV-A caused only a transient increase (Fig. 5).

DISCUSSION To our knowledge, this study is the first to investigate the photoprotective strategies in long-term acclimated nonsymbiotic sea anemone exposed to elevated UV-R levels. Our results clearly indicate that long-term photoacclimation can ameliorate cellular damage in A. tenebrosa exposed to UV-B. Molecules that can act as sunscreens, such as MAAs, are considered to play an important part in protecting corals (65) and sea anemones (31), from UV-B induced damage. Cnidarians living in shallow waters that have symbiotic zooxanthellae, are characterized by the presence of several MAAs at elevated levels (31,35,66). The concentrations of MAAs found in A. tenebrosa in this study were similar to those found in A. tenebrosa in Australian waters (9.05 nmol mg DW
1) (67), and in the aposymbiotic sea anemone Anthopleura elegantiss-

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Figure 3. Average (SE) oxidative damage in proteins (nmol mg protein
1) and lipids (nmol mg FW
1) in summer and winter collected Actinia tenebrosa exposed to PAR (P), PAR + UVA (PA), PAR + UVA + UVB (PAB) and PAR + UVA + UVB with UVB enhanced 20% (PAB-20%) radiation treatments for 4 days. Common letters indicate values that are not significantly different from one another using a multiple comparison test (Tukey’s HSD) at a significance level of 0.05.

ima (11 nmol mg DW
1) (68). A. tenebrosa, found in Otago Harbor contained three identified MAAs, shinorine, mycosporine 2-glycine and porphyra-334. A similar MAA composition was observed in both symbiotic and aposymbiotic An. elegantissima (35,68). In addition, these MAAs were shown to be important components of the MAA pool in the aposymbiotic sea anemones Aulactinia marplatensis and Oulactis muscosa (31). Actinia tenebrosa exhibited seasonal variation in total MAA levels, when sea anemones collected in summer and winter were compared, with lower total MAA levels observed in winter. The reduction of MAA levels in winter was due to lower shinorine and mycosporine 2-glycine levels (6% and 84%, respectively). Seasonal variation in MAA levels has been also identified in organisms including macroalgae (69), crustaceans (70) and cnidarians (45). These seasonal variations in MAA levels have been correlated with a seasonal change in UV-R exposure, which also occurs in the Otago harbor (70). As invertebrates lack the ability to synthesize MAAs, those that do not establish symbiotic associations with microalgae acquire MAAs entirely from a dietary source, e.g. Aplysia dactylomela (71), Strongylocentrotus droebachiensis (32), Evechinus chloroticus (72), E. superba (73) and Leptodiaptomus minutus (74). Thus, the level of MAAs in the food consumed determines the level of accumulation of MAAs within these invertebrates. It is possible, therefore, that the MAA levels found in A. tenebrosa, found in Otago Harbor, are also related to seasonal variations in MAA levels found in the food they consume. For example, the locally abundant euphausiid,

Nyctiphanes australis, which are likely consumed by A. tenebrosa, demonstrate a clear seasonal change in MAA concentrations (70). Previous studies (75–77) have shown that the accumulation of MAAs in invertebrates is not immediate and it takes from weeks to months to acquire sufficient MAA concentrations to effectively act a sunscreen, providing protection against UV-R. Hence, invertebrates require additional cellular mechanisms to reduce UV-R induced damage when exposed to UV-R prior to MAA accumulation. In this study while seasonal differences in MAA levels were observed, short-term exposure to UV-B did not change MAA levels. While some anthozoan species have been shown to possess genes encoding the enzymes required for the synthesis of the MAA Shinorine (78), it is generally accepted that in most invertebrates MAA levels can only be increased by the consumption of MAA containing organisms (79,80); hence, the lack of change in MAA levels observed in this study. Because of the photostability of MAAs (79,81) individuals collected in latesummer and those collected in the winter, could be exposed to artificial UV-R and the responses compared, to determine the importance of cellular mechanisms, other than sunscreens, for UV-R protection. Cellular mechanisms involved in the repair of damage caused directly by exposure to UV-R, such DNA damage in the form of CPDs, have been extensively studied in invertebrates (53,82,83). In this study, organisms sampled in summer exposed to artificially enhanced levels of UV-B and allowed to recover following exposure, showed no accumulation of CPDs over a

Photochemistry and Photobiology, 2014, 90

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Figure 4. Average concentrations (SE) of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPOX) and glutathione reductase (Gr) in seasonal UV-R-acclimated Actinia tenebrosa exposed to PAR (P), PAR + UVA (PA), PAR + UVA + UVB (PAB) and PAR + UVA + UVB with UVB enhanced 20% (PAB-20%) radiation treatments for 4 days. Common letters indicate values that are not significantly different from one another using a multiple comparison test (Tukey’s HSD) at a significance level of 0.05.

4-day experimental period. In contrast, organisms sampled in winter accumulated CPDs in a dose-dependent manner over a 4-day experimental period. These results suggest that high MAA levels, probably in combination with an increased capacity to repair direct UV-B induced DNA damage, can protect individuals from both the maximum ambient levels of UV-B exposure found in the Otago Harbor in summer and to elevated

levels of UV-B. Previous observations indicated that CPD accumulation in sea urchin larvae was mainly associated with MAAs levels and DNA repair rates (53). DNA repair, by photoreactivation, in the larvae of the tropical sea urchin Diadema setosum removes 90% of CPDs within of 4 h, while it takes 14 h to remove 90% of CPDs in the Antarctic species Sterechinus neumayeri, and approximately 24 h for complete removal (82). Such

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Figure 5. Average (SE) total glutathione, glutathione reduced (GSH) and glutathione oxidized (GSSG) in summer and winter collected Actinia tenebrosa exposed to PAR (P), PAR + UVA (PA), PAR + UVA + UVB (PAB) and PAR + UVA + UVB with UVB enhanced 20% (PAB-20%) radiation treatments for 4 days. Common letters indicate values that are not significantly different from one another using a multiple comparison test (Tukey’s HSD) at a significance level of 0.05.

differences in DNA repair in sea urchins are mainly attributed to metabolic changes associated with water temperature (82). In addition, in a recent study on coral planulae by Reef et al. (84) demonstrated the importance of photoreactivation for survival in the presence of high UV-R. They suggested that DNA repair combined with MAAs was sufficient to allow the survival of planulae despite exposure to high UV-R. The results discussed above confirm the importance of longterm accumulation of MAAs for UV-B protection, but how important are antioxidant defenses and protection against indirect oxidative damage in late-summer individuals, compared to winter individuals? Sea anemones sampled in summer and exposed to artificial UV-B at maximal ambient levels showed only a small increase in oxidative protein damage at the end of the 4-day experimental period, while those exposed to elevated UV-B had high levels of oxidative damage to both protein and lipids. In contrast, individuals sampled during winter showed greater oxidative damage to both proteins and lipids in response to all three UV-R treat-

ments (PA, PAB and PAB-20%), than summer sampled individuals. These results indicate increased sensitivity not only to UV-B, but also to UV-A-induced oxidative damage to macromolecules (26,28). Elevated levels of oxidative damage to DNA (8OHdG) have been observed in the larvae of the sea urchin S. droebachiensis exposed for prolonged periods to UV-A (28). These results suggest that oxidative damage in summer-acclimated individuals is unlikely to have a significant impact on animal growth and development under field conditions when individuals are fully acclimated to summer environmental conditions. However, the possibility that increased sensitivity to UV-R exposure exists during specific developmental windows cannot be ruled out. For example, Aranda et al. (85) demonstrated that in coral larvae some key developmental events are much more sensitive to UV-R than others. Surprisingly, sea anemones sampled in summer had only slightly higher levels of antioxidant defenses than those sampled in winter, prior to exposure to artificial UV-R, and would there-

Photochemistry and Photobiology, 2014, 90 fore appear to have only a slightly greater initial capacity to cope with oxidative stress than individuals sampled in winter, and acclimated to winter environmental conditions. Interestingly, of the antioxidant defense mechanisms investigated in this study only GPOX and glutathione were significantly influenced by artificial UV-B exposure, in both summer and winter-acclimated A. tenebrosa. Both GPOX and glutathione have a key role in H2O2 detoxification in marine invertebrates (86) as GPOX catalyzes the reaction between GSH and H2O2 to generate GSSH + H2O (87). A previous study on the sea anemone Nematostella vectensis indicated that they have a range of antioxidant enzymes including 12 genes for GPOX and Gr (88). In A. tenebrosa it appears that GPOX could be the main ROS detoxification mechanism as only low levels of CAT activity were detected in this study. In other invertebrates where elevated GPOX levels are found CAT activity is often reduced or completely absent (86). It was notable that while winter sampled sea anemones showed increased dose sensitivity and earlier induction of antioxidant defense mechanisms than summer sampled individuals, in response to artificial UV-R, their capacity to up regulate their antioxidant defenses was similar. Hence, because of the absence of MAAs they suffered much more oxidative damage than summer sampled individuals. There have been several studies on the influence of UV-R on ROS generation, oxidative stress and antioxidant capacity in symbiotic cnidarians, such as corals, and sea anemones (23,24, 45,46,49). For example, seasonal variations in CAT and glutathione-S transferase (GST) have been observed in the shallow water coral Pocillopora capitata caused by increases in UV-R and in water temperature (45). In addition, increases in SOD activities, in response to UV-B, have been observed in the body column tissues of Anemonia sulcata and Anemonia rustica (24). Increased GPOX activity has also been observed in symbiotic sea anemones (24) and corals (45), but the role of UV-R as a regulator of GPOX in corals is inconclusive (46). To our knowledge this study is the first to investigate changes in oxidative stress and antioxidant metabolism in a nonsymbiotic sea anemone exposed to UV-R and to address the relative importance of long-term versus short-term acclimation responses. Our results suggest that GPOX and glutathione may play a key role in protecting A. tenebrosa from oxidative stress. Evidence for the importance of GPOX as a mechanism to reduce ROS levels in A. tenebrosa comes not only from the increased activities observed, but also from the observations that GSH levels show a transient drop and GSSG levels a transient increase in response to elevated UV-B exposure, followed by an increase in GSH and total glutathione levels. GPOX requires glutathione as a source of reducing potential and hence increased glutathione levels, and/ or recycling, would be required to support the antioxidant function of this enzyme (89). The results of this study also clearly show that A. tenebrosa has only a limited capacity to increase antioxidant metabolism in response to UV-R exposure and that this modest increase in antioxidant defenses cannot substitute for MAA accumulation as a means of UV-R protection from both direct and indirect cellular damage. As it has been suggested that MAAs can function both as sunscreens and antioxidants (90), so it is possible that A. tenebrosa has evolved to rely on the accumulation of MAAs as a means of supplementing the conventional antioxidant systems measured in this study.

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While this study has clearly shown that in A. tenebrosa UV-R causes oxidative damage and induces an increase in the sea anemone antioxidant defense mechanisms, the degrees of which are related to MAA levels and seasonal acclimation, it is also possible that a major contributor to the relatively small changes observed in this study could be related to the behavioral responses of this organism to UV-R. It is important to highlight that during the course of our experiments A. tenebrosa tended to contract their oral disks, as has been observed previously in A. elegantissima (23), possibly as a mechanism to minimize UVR induced ROS generation and thereby reduce the energetic costs associated with defense and cellular repair. Acknowledegments—This research was funded by the University of Otago through the PhD International Research Scholarship. Financial support in the form of the University of Otago Publication bursary was also provided to V.M.C.

SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article: Figure S1. Spectral transmission profiles for the filters used to control UV-R exposures. Figure S2. HPLC/MS profiles of shinorine (A), mycosporine 2-glycine (B) and porphyra-334 (C) in Actinia tenebrosa.

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