Natural high pCO2 increases autotrophy in Anemonia viridis (Anthozoa) as revealed from stable isotope (C, N) analysis

Contemporary cnidarian-algae symbioses are challenged by increasing CO2 concentrations (ocean warming and acidification) affecting organisms' biological performance. We examined the natural variability of carbon and nitrogen isotopes in the symbiotic sea anemone Anemonia viridis to investigate dietary shifts (autotrophy/heterotrophy) along a natural pCO2 gradient at the island of Vulcano, Italy. δ13C values for both algal symbionts (Symbiodinium) and host tissue of A. viridis became significantly lighter with increasing seawater pCO2. Together with a decrease in the difference between δ13C values of both fractions at the higher pCO2 sites, these results indicate there is a greater net autotrophic input to the A. viridis carbon budget under high pCO2 conditions. δ15N values and C/N ratios did not change in Symbiodinium and host tissue along the pCO2 gradient. Additional physiological parameters revealed anemone protein and Symbiodinium chlorophyll a remained unaltered among sites. Symbiodinium density was similar among sites yet their mitotic index increased in anemones under elevated pCO2. Overall, our findings show that A. viridis is characterized by a higher autotrophic/heterotrophic ratio as pCO2 increases. The unique trophic flexibility of this species may give it a competitive advantage and enable its potential acclimation and ecological success in the future under increased ocean acidification.

I ncreasing carbon dioxide (CO 2 ) emissions drive ongoing ocean acidification (OA) and place marine ecosystems in a vulnerable state 1 . Predictions warn of a further decrease of 0.3-0.5 pH units in oceanic surface water by the end of this century 2 . Natural CO 2 vents at sub-tropical coastal areas [3][4][5] and tropical reefs 6 serve as natural laboratory locations to study long-term effects of elevated pCO 2 (pH) across many biological and spatial scales. Such a location has been reported in the Levante Bay of Vulcano Island (Italy) in the Mediterranean Sea where many studies have examined physiological adaptations of biota to OA, including seagrass 7 , benthic micro-and macroalgaes 8,9 , sea urchins 10 , and sea anemones 11,12 . The distinctive characteristics of this location render it a unique environmental setting where the seawater chemistry varies along a pCO 2 gradient of several hundred meters moving away from the venting source. The submarine gas emissions in Levante Bay are characterized by high CO 2 content volume (.90%) and variable low H 2 S (ranging 0.8 to 2.5% volume) 13 .
A large body of research has focused on the potential impact of OA on reef organisms, particularly scleractinian corals. However, non-calcifying cnidarians such as sea anemones have received less attention 14 . Like many cnidarians, they are mixotrophic organisms, which derive their energy from both photoassimilates translocated from the dinoflagellate symbionts (Symbiodinium) and from a variety of external food sources 15 . Symbiodinium utilize bicarbonate (HCO 3 2 ), rather than CO 2(aq) , as the primary source for photosynthesis 16 . Extrinsic sources of carbon for the host include zooplankton and particulate organic carbon (POC) 17 . The two partners that make up the holobiont interact at the basic metabolic level, which includes reciprocal fluxes of energy and nutrient-rich compounds 18 . Anemonia viridis Forskål (Cnidaria: Anthozoa), the temperate Mediterranean species chosen for this study, occurs naturally at high densities throughout Levante Bay and harbors the dinoflagellate Symbiodinium muscatinei LaJeunesse and Trench (Dinomastigota: Dinophyceae) 12 . Hence it is a powerful comparative model to assess the effects of the changing seawater environment along a natural pCO 2 gradient.
Other reports on the response of A. viridis near CO 2 vents discovered changes in their associated microbial communities 19 , reduced dimethylsulfoniopropionate (DMSP) production 12 and enhanced productivity 3,11 .
The purpose of this paper is to investigate dietary changes of A. viridis using isotopic compositions, particularly carbon source shifts in the anemone metabolism, in response to high pCO 2 /low pH conditions in situ. We measured how the natural variability of carbon and nitrogen isotopes in Symbiodinium and host tissues of A. viridis varies along a natural pCO 2 gradient. This was compared with other key physiological parameters (i.e. total protein concentration; Symbiodinium density, mitotic index, and chlorophyll concentration) which were used in the present and in previous studies 11 . Since the d 13 C and d 15 N signatures of an organism are related to those of its diet 20-24 , our main objective was to estimate the relative contribution of photosynthetic compounds versus heterotrophically derived food to the anemone energetic budget (autotrophic/heterotrophic ratio) with increasing seawater pCO 2 . This may facilitate better understanding of the environmental fate of cnidarians in a high CO 2 world.

Results
Visual observations made during the course of sampling found anemones at all sampling sites attached to hard substratum at high abundances (of ca. 10-40 anemones m 22 ), consistent with previous findings 11 . Anemones appeared to be healthy with their tentacles fully extended and no visible excess amounts of mucus at the high pCO 2 site (Fig. 1b). Data for seawater pH, pCO 2 , TA, temperature and light intensity at all anemone sampling sites is summarized in Figure 1a.

Discussion
A. viridis collected at all pCO 2 sites lacked any apparent signs of stress (i.e. no mucus, tentacles fully extended; see Fig. 1b). Their general health was further supported by our results for physiological and algal characteristics. Protein concentrations, which are widely accepted as a sensitive indicator for the health of an organism 25 , showed no difference between sampling sites, indicating A. viridis was in fact well acclimated to the high seawater pCO 2 (Fig. 2a). In addition, there were no changes in Symbiodinium densities and their chlorophyll a concentrations along the pCO 2 gradient (Fig. 2b). This is in agreement with observations of the anemone Anthopleura elegantissima, following exposure to elevated pCO 2 conditions in a laboratory setting, using the standard algal cell normalization to mg of protein methodology as in the present study 14 . However, Symbiodinium densities in A. viridis under high pCO 2 conditions nearby the vent at Vulcano have been reported to increase relative to algal densities in anemones at the control site 11 . This discrepancy may be the result of a different methodology (using surface area as a normalization index in the same study 11 ) in determining algal cell densities. The handling of anemones greatly influences tentacle contraction, which may have led to inaccuracy in surface area measurement, thereby making the comparison of results difficult.
The substantial increase in dividing algal cells under elevated pCO 2 (MI; Fig. 2c) is in accordance with previous studies reporting high MIs in anemones under high pCO 2 11,14 . It is important to note that there was no variation in algal genotype as the anemones from all three sites were found to harbor Symbiodinium type A19 12 , excluding the possibility that genetic makeup of the Symbiodinium is responsible for the difference. The marked increase in algal division is most likely a direct result of massive CO 2 input, as Symbiodinium in anemones remain carbon limited under normal conditions 11,14,26,27 . Since cnidarians are required to maintain cell-specific densities of their algal symbionts to avoid toxicity from excess oxidative products 28 , the host may initiate either active expulsion of symbionts and/or chemically-signaled arrest of algal reproduction 29 . Here, the high MIs but same algal densities, relative to algal densities at the control site, suggest that the anemones were unable to regulate algal repro-     duction under the elevated pCO 2 conditions and therefore densities were likely maintained through Symbiodinium expulsion.
Considering that in addition iron (Fe) is the most important trace element for algal growth 30 , Fe enrichment in the seawater near the vent site 13,31 may have also affected algal proliferation to some extent.
The acidification of seawater close to the venting source arises from the constant gas emissions 13 . In addition to total DIC increasing by 17% at the high pCO 2 site as compared to the control, CO 2(aq) increased near the venting source (7-fold increase at the high pCO 2 site; see Table 1). Although the carbonate system still consists mostly of bicarbonate (94%), CO 2(aq) increased from less than 1% at the control site to 4% at the high pCO 2 site (Table 1). Nonetheless, the isotopic composition of the inorganic carbon source in this area for the anemones appears to be constant as data shows that d 13 C DIC does not change between sites (Fig. 3). Consequently, the pronounced and persistent depletion in 13 C in the tissues of A. viridis and its Symbiodinium close to the vent cannot be explained by the assimilation of a 13 C-depleted carbon source. The large increase in pCO 2 in the seawater (Table 1; Fig. 1a) and its availability for A. viridis most likely account for the decrease in A. viridis d 13 C values in both Symbiodinium and host tissue. The values near the vent (Fig. 4a, b) were well below the lower limit of the range reported previously for both tropical and subtropical sea anemones and Symbiodinium 32, 33 . d 13 C T values decreased at the intermediate and high pCO 2 sites to 217.62 6 0.19% and 219.12 6 0.16%, respectively, as compared to the control site (216.66 6 0.2%) (Fig. 4a), suggesting an increase in photosynthetically fixed carbon relative to heterotrophically acquired carbon in the host 20,34,35 . Taking seasonal and regional variability into account, average zooplankton and particulate organic carbon (POC) d 13 C values reported in the area for surface waters range between 221 and 222% 36 . We assumed that the availability of these extrinsic carbon sources was constant across all sampling sites in our study, as the relatively short distance between sampling sites (,500 m) and their orientation in Levante Bay towards the open sea renders differences in food availability most unlikely as a factor. Based on mass balance estimation, our calculations show about 5% heterotrophic input to d 13 C T at the control site (using d 13 C T 5 216.66% and d 13 C S 5 215.1%, assuming   d 13 C zooplankton/POC 5 222%). This is typical of cnidarian-algae symbioses, in which Symbiodinium may contribute up to 95% of their photosynthetically-produced carbon to the host 37 . Based on the same assumptions, at the high pCO 2 site the heterotrophic input to d 13 C T reduced to about 2.5% (using d 13 C T 5 219.12% and d 13 C S 5 218.21%, assuming d 13 C zooplankton/POC 5 222%), leading to a greater autotrophic input. This observation is also supported by the difference in d 13 C values between host tissue and Symbiodinium, which reflects the relative contribution of heterotrophy and photosynthesis to fixed carbon 20,38 . Cnidarian host tissue and Symbiodinium stable carbon isotopic values are usually within 2% of each other 20,39,40 . There was a significant reduction in d 13 C S -d 13 C T with increasing pCO 2 from 1.56 6 0.21% at the control site to 0.96 6 0.31% and 0.9 6 0.17% at the intermediate pCO 2 and high pCO 2 sites, respectively (Fig. 4a). This further indicates an increase in the autotrophic/heterotrophic ratio via translocated autotrophic carbon to the host.
Our results suggest that elevated pCO 2 near the vent promotes carbon isotope fractionation by Symbiodinium during photosynthesis, leading to lighter d 13 C S values. d 13 C S showed a substantial decrease from 215.1 6 0.28% at the control site to 216.65 6 0.37% and 218.21 6 0.24% at the intermediate and high pCO 2 sites, respectively (Fig. 4a). Many studies have shown that d 13 C is depleted in marine photosynthetic organisms under elevated   [41][42][43][44] . Under normal conditions, the majority of Symbiodinium carbon requirements (,85%) are met via energy-demanding carbonconcentrating mechanisms (CCMs), whilst the remainder diffuses passively from seawater to the Symbiodinium cells 28 . When pCO 2 is elevated, CO 2(aq) can replace HCO 3 2 as the main carbon source for photosynthesis while energy-consuming CCMs become less important 43,45 . Form II ribulose 1,5-bisphosphate carboxylase/oxygenase (form II Rubisco), which is the carboxylating enzyme in Symbiodinium 46 , discriminates against 13 C 47 . Enhanced levels of pCO 2 in the proximity of the vent diffuse to the Rubisco, which favors 12 C for carbon fixation and ultimately results in a lightning trend of d 13 C S values. Krief et al. (2010) reported the same trend in two species of scleractinian corals after experimental exposure to high pCO 2 in a controlled pCO 2 system. While Krief et al. (2010) kept corals under elevated pCO 2 for a period of 14 months, our in situ study at the CO 2 vent site lends insight into a long-term exposure scenario 48 . d 15 N T and d 15 N S values did not change along the pCO 2 gradient, suggesting that the anemones' function and performance reside within normal bounds close to the vent after long-term exposure to acidification conditions (Fig. 5a). Further supporting this concept is the lack of change in C/N ratio between sites (Fig. 5b). The C/N ratio is considered a good proxy for an organism's condition since it reflects the ratio of lipids and carbohydrates to proteins 49 . The apparent absence of preferential accumulation/loss of lipids, carbohydrates or proteins in A. viridis in high pCO 2 /low pH surroundings indicates therefore that the anemones were well acclimated.
Generally, animals exposed to high pCO 2 /low pH have to compensate for acid-base imbalance in intra-and extracellular spaces thereby imposing elevated metabolic costs 50 . A recent study by Laurent et al. (2014) demonstrated the high capacity of A. viridis to regulate against decreases in internal and external pH, thereby maintaining normal cellular metabolism and physiology 51 . Our results indicate the adaptation and potential resilience of A. viridis to acidification conditions, as physiological data (i.e. protein content, Symbiodinium density and chlorophyll a concentration; Fig. 2a, b), along with d 15 N values and C/N ratios (Fig. 5a, b), remained unaffected among sites along the pCO 2 gradient. Moreover, the high pCO 2 environment probably stimulated cell division of algal symbionts (Fig. 2c).
We have shown that the anemone host relies more on photosynthetically derived carbon under elevated pCO 2 . We propose that A. viridis optimizes energy utilization under elevated pCO 2 through an increased autotrophic input, although isotopic data show that heterotrophy is maintained as an additional source of energy/nutrients. These factors may contribute, at least in part, to the increased size and abundance of the A. viridis population proximate to the vent site as reported in a previous study 11 . In conclusion, increased autotrophic/heterotrophic ratio may enhance the competitive advantage of symbiotic anemones over other invertebrates and improve their ecological success in benthic communities. These are valuable findings that merit further study for predicting the performance of noncalcifying symbiotic cnidarians in future high-CO 2 oceans.

Methods
Study sites. This study was conducted along the sublittoral in Levante Bay, Vulcano  Island (38u 259 N, 14u 579 E), part of the Aeolian Island chain, NE Sicily (Fig. 1a) in May 2012. Shallow-water CO 2 vents create a natural pCO 2 /pH gradient along the north-easterly side of the bay, ranging from pH 6.05 to 8.29 at .350 m from the vent site 8,13 .
Three sites were selected for animal sampling in accordance with previous studies (see Fig. 1a) 7,8,11,13 . Site 1 (control) was an ambient seawater reference station, located outside the vent area (.400 m); Site 2 (intermediate pCO 2 ) was ,300 m away from the CO 2 vents; Site 3 (high pCO 2 ) was in the proximity of the CO 2 vents (,260 m). Sampling at the primary vent site (indicated by the star symbol in Fig. 1a) was for collection of seawater samples only.
Carbonate chemistry and physical measurements. Seawater pH (NBS scale) and temperature were measured at all sites several times a day for 4 days using a pH meter (YSI Professional Plus, Handheld Multiparameter Instrument, USA). Water samples for total alkalinity (TA) analysis were collected from each site, cooled and stored in the dark until analysis. TA was quantified with a Metrohm 862 compact titrosampler 52 Table 1. Light intensity at each site was measured hourly for 3 consecutive days close to the seabed (1-2 m depth) with HOBO PendantH Temperature/Light data loggers (Onset, Pocasset, MA, USA). The logged light data were converted from lux to mmol quanta m 2 s 21 (Fig. 1a) 55 .
Sample collection in the field. Anemones. A. viridis, a dominant benthic organism in Levante Bay, was prevalent throughout the study area. Sixteen anemones were collected randomly from each site at a depth of 1-2 m and immediately frozen until further analyses. To minimize any confounding responses due to age and/or size all samples were of similar size (oral disc diameter of 2.5-3.5 cm) 56 . Between 5 and 10 tentacles were clipped from each anemone at every site (n 5 16). Tentacles were processed for total protein and algal characteristics (i.e., Symbiodinium density, chlorophyll a concentration and mitotic index) at the sampling site. Samples were weighed (CT 1202, Citizen, accuracy 0.01 g) and homogenized in 0.2 mm sterile filtered seawater (FSW) with an electric homogenizer (DIAX 100 homogenizer Heidolph Instruments GmbH & Co. KG, Schwabach, Germany). The homogenate and all anemones were immediately frozen and then transported on dry ice to the Interuniversity Institute for Marine Sciences (IUI), Israel, where they were stored at 280 uC pending analyses.
Seawater. Seawater samples were collected from the four sites for carbon isotopes of dissolved inorganic carbon (DIC; d 13 C DIC ) and oxygen isotopic analysis (d 18 O seawater ). Triplicate samples for d 13 C DIC analysis were immediately poisoned upon collection with 60 ml saturated solution of mercuric chloride and stored in 60 ml brown bottles at room temperature until analysis. Triplicate samples for d 18 O seawater analysis were collected in 50 ml test tubes (Stardest) and stored at room temperature until analysis.
Total protein, Symbiodinium density, mitotic index and chlorophyll concentration. The tissue homogenate of each anemone (n 5 16) was further processed and analyzed for measurements of physiological parameters. Total protein analysis was performed by removing 100 ml of the tissue homogenate and sonication on ice with a Branson Sonifier B12 (Branson Sonic Power Co., Danbury, Connecticut, USA) for 20 s. Quantification was done after Bradford (1976) 57 . Optical density was read at 595 nm using an ELISA reader (Multiskan spectrum, Thermo Fisher Scientific Inc., USA).
For measurement of algal characteristics, 2 ml of homogenate of each sample (n 5 16) were centrifuged (5000 rpm at 4uC; 4K15 centrifuge, Sigma) and re-suspended four times in FSW. Re-suspended Symbiodinium were used for chlorophyll a extraction in acetone (100%) at 4uC in the dark for 24 hours. Concentrations of chlorophyll a were measured using spectrophotometry (Ultrospec 2100 pro, GE Bioscience, USA) and calculated using standard equations 58 . Chlorophyll concentration was calculated per Symbiodinium cell. Symbiodinium densities were quantified from 4 replicate counts using a Neubauer haemocytometer and normalized to protein concentration. Mitotic index (MI) was measured as an indicator of Symbiodinium growth and was calculated as a percentage of doublets with a complete cleavage furrow observed per 1000 cells (n 5 8 per sampling station) 59 . Separation of anemone tissue and Symbiodinium for isotope analysis. Subsamples of 250 mg were excised from the tentacles of each anemone (n 5 5 per site) and placed in sterile 15 ml falcon tubes (Stardest). After adding 1 ml 0.2 mm filtered seawater (FSW), an electric homogenizer (DIAX 100 homogenizer Heidolph Instruments GmbH & Co. KG, Schwabach, Germany) was used to homogenize the tissue extract for 2 min. Separation of anemone tissue and Symbiodinium was done by the following protocol. The homogenate was centrifuged for 5 min at 5000 rpm (4K15 centrifuge, Sigma) to separate the algae (pellet) and the host tissue (supernatant). Visual inspections revealed no crossover of material between these components, but both were washed carefully. The host supernatant was homogenized and centrifuged for 10 min at 13,500 rpm (4K15 centrifuge, Sigma, USA), resulting in pelleted host material for analysis. The Symbiodinium pellet was then re-suspended in 1 ml FSW, homogenized, and centrifuged for 5 min at 5000 rpm (4K15 centrifuge, Sigma, USA). The procedure was repeated twice in order to remove remaining tissue. All samples were washed with double-distilled water (DDW) to remove any remaining salts. Both the host tissue and Symbiodinium samples were dried with a lyophilizer (VirTis, Sentry 2.0, SP Scientific, USA) for 24 h for further isotopic analysis.
Stable isotope analyses. The isotopic measurements were made at the stable isotopes laboratory in the Department of Earth and Planetary Sciences, the Weizmann Institute of Science, Israel. The oxygen, carbon and nitrogen isotope measurements are reported in the conventional d-notation.
Anemone tissue and Symbiodinium samples. d 13 C and d 15 N of 240-270 mg of dried tissue and algae were analyzed using an elemental analyzer (CE 1110) interfaced to the MAT 252 mass spectrometer. Long term precision of working standards for d 13 C is 0.05% and for d 15 N is 0.1% relative to V-PDB and Air respectively (61s SD). The carbon to nitrogen ratios (C/N) of anemone tissue and Symbiodinium were calculated from simultaneous %C and %N.
Seawater samples. d 18 O seawater was analyzed by equilibrating 0.5 ml of samples with a mixture of 0.5% CO 2 in He at 25 uC for 24 h. The samples were analyzed on a Gas Bench II connected in-line to a Finigan MAT 252 mass spectrometer. The results are reported relative to VSMOW with 0.08% (61s SD) long-term precision of the laboratory working standards.
For d 13 C DIC analysis, 1 ml sea water was injected into vials, flushed with He gas, acidified with 0.15 ml orthophosphoric acid (H 3 PO 4 ) and left to react for 24 h at 25 uC. The samples were analyzed on a Gas Bench II and Finigan MAT 252. The results are reported relative to VPDB with 0.08% long-term precision (61s SD) of the NaHCO 3 laboratory standard.
Data analyses. All data was checked for normality using the Kolmogorov-Smirnov test and for homogeneity of variance using Cochran's test. In cases in which homogeneity of variance was achieved, we used one-way ANOVA and a multiple comparison test (Tukey). If homogeneity of variance or normality was not achieved, we used a non-parametric Kruskal-Wallis ANOVA and post-hoc Mann-Whitney Utests for separation of significant factors. Differences between factors were considered significant for a P value , 0.05. Unless otherwise specified, mean values are presented 6 standard error of mean (SEM). All data were analyzed using SPSS version 20 (SPSS IBM, New York, USA).