Global Change Biology (2014) 20, 765–777, doi: 10.1111/gcb.12478

Habitat traits and food availability determine the response of marine invertebrates to ocean acidification C H R I S T I A N P A N S C H 1 , 2 , I R I S S C H A U B 1 , 3 , J O N A T H A N H A V E N H A N D 2 and M A R T I N W A H L 1 1 Department of Marine Ecology, GEOMAR Helmholtz Centre for Ocean Research Kiel, Kiel 24105, Germany, 2Department of Biological and Environmental Sciences – Tja¨rno¨, University of Gothenburg, Tja¨rno¨, Stro¨mstad 45296, Sweden, 3Institute of Biological Sciences, Marine Biology, University of Rostock, Rostock 18059, Germany

Abstract Energy availability and local adaptation are major components in mediating the effects of ocean acidification (OA) on marine species. In a long-term study, we investigated the effects of food availability and elevated pCO2 (ca. 400, 1000 and 3000 latm) on growth of newly settled Amphibalanus (Balanus) improvisus to reproduction, and on their offspring. We also compared two different populations, which were presumed to differ in their sensitivity to pCO2 due to differing habitat conditions: Kiel Fjord, Germany (Western Baltic Sea) with naturally strong pCO2 fluctuations, and the Tja¨rno¨ Archipelago, Sweden (Skagerrak) with far lower fluctuations. Over 20 weeks, survival, growth, reproduction and shell strength of Kiel barnacles were all unaffected by elevated pCO2, regardless of food availability. Moulting frequency and shell corrosion increased with increasing pCO2 in adults. Larval development and juvenile growth of the F1 generation were tolerant to increased pCO2, irrespective of parental treatment. In contrast, elevated pCO2 had a strong negative impact on survival of Tja¨rno¨ barnacles. Specimens from this population were able to withstand moderate levels of elevated pCO2 over 5 weeks when food was plentiful but showed reduced growth under food limitation. Severe levels of elevated pCO2 negatively impacted growth of Tja¨rno¨ barnacles in both food treatments. We demonstrate a conspicuously higher tolerance to elevated pCO2 in Kiel barnacles than in Tja¨rno¨ barnacles. This tolerance was carried over from adults to their offspring. Our findings indicate that populations from fluctuating pCO2 environments are more tolerant to elevated pCO2 than populations from more stable pCO2 habitats. We furthermore provide evidence that energy availability can mediate the ability of barnacles to withstand moderate CO2 stress. Considering the high tolerance of Kiel specimens and the possibility to adapt over many generations, near future OA alone does not seem to present a major threat for A. improvisus. Keywords: adaptation, Amphibalanus (Balanus) improvisus, barnacles, calcification, carry-over effects, energy availability, eutrophication, global change, naturally acidified ecosystem, ocean acidification Received 14 August 2013; revised version received 31 October 2013 and accepted 8 November 2013

Introduction Anthropogenically increased atmospheric CO2 causes a reduction in oceanic pH – a phenomenon referred to as Ocean Acidification (‘OA’; Gattuso & Hansson, 2011; Caldeira & Wickett, 2003). While oceanic pH decreases at relatively constant rates of ca. 0.002 units per year (Doney et al., 2009), the progress of OA in coastal habitats is much more variable (Duarte et al., 2013). Future scenarios of OA for open-ocean waters also do not reflect patterns described for most coastal areas (Wootton et al., 2008; Feely et al., 2010; Melzner et al., 2013). In coastal regions, river runoff, upwelling, eutrophication and biological activity modify the effects of atmospherically driven OA (Duarte et al., 2013). In many highly productive areas, this leads to severe Correspondence: Christian Pansch, tel. +46 317869679, fax +46 317861333, e-mail: [email protected]

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fluctuations in the carbonate system at frequencies from hours to months and produces local levels of acidification that can greatly exceed average projections for the end of this century (Caldeira & Wickett, 2003; Wootton et al., 2008; Miller et al., 2009; Feely et al., 2010; Duarte et al., 2013). Most projections to date, however, have only been made for open-ocean waters or rather un-impacted coastal regions. In the Western Baltic Sea, and especially in coastal bays such as the Kiel Fjord and the Eckernfo¨rde Bay, annual mean surface seawater pCO2 is typically ca. 800 latm, and peak values of 2400 latm occur during upwelling events (Thomsen et al., 2010, 2013; Melzner et al., 2013; Saderne et al., 2013). Given the pronounced differences in mean and fluctuation (amplitude and frequency) with regard to the carbonate system and seawater pCO2, we might expect that coastal and oceanic species as well as species from un-impacted and impacted coastal regions differ in their sensitivity to acidification (e.g., Duarte 765

766 C . P A N S C H et al. et al., 2013). Understanding how organisms from naturally acidified environments in marginal seas cope with OA may indicate the species’ potential for adaptation to this aspect of global change. The impacts of stressors, such as elevated pCO2, on organisms are neither simple nor uniform. They are highly species specific and are modulated by numerous factors such as the stress intensity, the duration and fluctuations of the stressor, the life stage, the stress history of the individual, the interactions among synchronous stressors, the existence of compensating mechanisms, the genetic potential for adaptation (i.e., standing genetic variation) or phenotypic plasticity, and the organism’s energy demand balanced against energy availability in the habitat (Kroeker et al., 2010, 2013; Wahl et al., 2011; Whiteley, 2011; Wong et al., 2011; Duarte et al., 2013; Harvey et al., 2013). Thus, organisms living in stable, but oligotrophic, habitats might be sensitive to increases in pCO2, whereas organisms from fluctuating (often eutrophic) habitats might be more tolerant to elevated pCO2 (Findlay et al., 2009, 2010a,b; Findlay, Burrows et al., 2010; Dupont et al., 2010; Whiteley, 2011). Available evidence supports these assertions: mussels living in eutrophic food rich habitats were able to withstand naturally high pCO2 (Thomsen et al., 2010, 2013; Melzner et al., 2011), whereas mussels in less eutrophic habitats suffered at similar pCO2 (Hall-Spencer et al., 2008; Rodolfo-Metalpa et al., 2011). Almost all projections of the effects of OA on marine ecosystems assume that responses measured in present-day populations, often in short-term (small fraction of a species’ life span) experiments, will apply to future populations. Short-term – and even long-term (larger fraction of a species’ life span up to its entire life cycle) but within-generation responses to environmental shifts – mainly reflect physiological acclimation capacities, that is, plasticity of individual organisms (Pigliucci, 2001). In contrast, only trans-generational responses reflect the ecological and evolutionary impacts of environmental shifts and can allow quantification of the capacity of populations to adapt to a changing environment (Sunday et al., 2011). Despite the importance of carry-over effects for adaptation to future OA, few studies have considered links between life-history stages (McDonald et al., 2009), or between parents and their offspring (Miller et al., 2012; Parker et al., 2012) when determining a species’ response to elevated pCO2. Here, we examined different performance metrics (survival, growth, calcification, moulting frequency, reproduction and shell strength) in 2-week-old juveniles to adults of the ecologically important barnacle Amphibalanus improvisus under food-replete and

food-restricted conditions in ambient, moderate and high pCO2. We also determined whether the effects of elevated pCO2 were carried-over from parents to their offspring by determining F1 larval development and juvenile growth. We furthermore compared the sensitivity of two populations of barnacles to elevated pCO2 from a naturally pCO2-variable (Kiel Fjord, western Baltic Sea) and a more pCO2-stable location (Tja¨rno¨, eastern Skagerrak). We hypothesized (i) that food availability will affect barnacle sensitivity to elevated pCO2; (ii) that stress impacts will be carried over to the offspring generation and to subsequent life-history stages; and (iii) that barnacle populations from fluctuating pCO2 environments are more tolerant to elevated pCO2 than barnacles from more stable habitats.

Materials and methods Amphibalanus improvisus can be found worldwide in oceanic and brackish waters and is by far the most common barnacle species in the Western Baltic Sea (Foster, 1987). Recruitment takes place mainly during summer and early autumn (Berntsson & Jonsson, 2003; Pansch et al., 2012). Eggs are fertilized internally and develop within the mantle cavity before free-swimming Stage-I nauplius larvae hatch (Thiyagarajan et al., 2003). Nauplii pass through six stages before they metamorphose into a non-feeding cypris larva (Jones & Crisp, 1954), which settles and metamorphoses to the juvenile barnacle. All experiments were conducted at the GEOMAR Helmholtz Centre for Ocean Research – Kiel, Germany and at the Sven Lov
en Centre for Marine Sciences – Tja¨rno¨, Sweden during summer and autumn 2011. Juvenile barnacles (Amphibalanus improvisus) were collected on transparent settlement panels (Perspexâ, 9 9 9 cm; Adolf Richter, Kiel, Germany) from the subtidal zone during the peak settlement season in the inner Kiel Fjord (54°19.5′N, 10°09.0′E, mid-June) and the Tja¨rno¨ archipelago (58°52.5′N, 11°08.1′E, mid-August). Panels were submerged horizontally at 1.5 m depths for 2 weeks. Settlement was restricted to the under side of the Perspex panel by covering the topside with a grey PVC panel. After retrieval from the field, barnacle density was standardized (26 individuals per panel) and dispersion maximized, by gently removing surplus barnacles. Two settlement panels were distributed to each aquarium of the different food (two levels) 9 acidification (three levels) treatment combinations (see below). Treatment combinations were replicated eight times (aquaria) for the Kiel experiment and six times for the Tja¨rno¨ experiment. A full set of eight and six panels was left in the field at each location, respectively, to observe in situ performance of the barnacles.

Food and acidification treatment levels Newly settled barnacles were reared following common barnacle culture methods (Thiyagarajan, 2010). After retrieval © 2013 John Wiley & Sons Ltd, Global Change Biology, 20, 765–777

H A B I T A T A N D F O O D D E T E R M I N E O A S T R E S S R E S P O N S E 767 from the field, barnacles were fed daily with a 1 : 1 mixture of Chaetoceros calcitrans and Skeletonema costatum. From week three onwards, barnacles were additionally fed with two-dayold brine shrimp nauplii (Artemia salina). From week five onwards, barnacles were fed brine shrimp only. Every 2 weeks, food concentrations were increased (Table S1), and the number of barnacles on the panels was haphazardly reduced to compensate for barnacle growth and increasing food consumption. The amount of food (number of algal cells or Artemia) added to ‘high-food’ treatments was five times that added to ‘low-food’ treatments. This 5 : 1 ratio was maintained during the entire experiment. Microalgae were cultured in filtered (0.2 lm) and autoclaved seawater enriched with f/2 medium. Algal densities were determined using a haemocytometer (Neubauer, Marienfeld, Germany) under a microscope. Artemia nauplii were cultured in filtered seawater in aerated 1-l plastic bottles, and densities were determined under a stereomicroscope. Three acidification levels were chosen to reflect the high natural variability of pCO2 in coastal seas combined with future scenarios (Thomsen et al., 2010, 2013; Wahl et al., 2011; Melzner et al., 2013). In Kiel, acidification was achieved by direct aeration of the experimental aquaria with either ambient air (‘ambient’) or premixed gas with pCO2 concentrations of 1120 ppm (‘moderate’) or 4000 ppm (‘high’). In Tja¨rno¨, acidification was achieved by direct aeration of the experimental aquaria with either ambient air (‘ambient’) or air enriched with pure gaseous CO2 controlled by computerized pH controllers (NBS scale, resolution: 0.01 pH units; Aqua Medic, Bissendorf, Germany; IKS, Karlsbad, Germany). Target pH values for the Tja¨rno¨ experiment were determined by routinely equilibrating experimental seawater with ambient air or custom mixed-gases (AGA Gas AB, Enko¨ping, Sweden) at 977 ppm (‘moderate’) and 3000 ppm CO2 (‘high’). The achieved pH levels were then used to set the respective pH controllers.

Experimental setup and carbonate chemistry measurements For both experiments, panels were held in small (3.7 l) covered plastic aquaria supplied with flow-through seawater pumped directly from the field. Seawater was pumped into header tanks in constant temperature rooms (20 °C) from which each aquarium was individually supplied (flow rate ca. 1.5 l h 1). In the Kiel experiment, juveniles (initial mean size (SD) 0.794  0.079 mm) were maintained under these treatment combinations for 20 weeks. In the Tja¨rno¨ experiment, juveniles (initial mean size 1.205  0.100 mm) were maintained for 5 weeks. Both experiments used a 12 : 12 h day/night cycle. Temperature and pHNBS (pH at the NBS scale) were maintained by thermostat and pH-stat (see above) but were additionally measured twice per week in four replicates in Kiel and in all six replicates in Tja¨rno¨ using a WTWâ (Weilheim, Germany) 330i pH meter equipped with a SenTixâ (Weilheim, Germany) 81 pH electrode, and a WTWâ Cond 340i equipped with a TetraConâ (Weilheim, Germany) 325 salinity electrode

© 2013 John Wiley & Sons Ltd, Global Change Biology, 20, 765–777

(Kiel) or a YSI 30â (Brannum Lane, Yellow Springs, OH, USA) multiprobe (Tja¨rno¨). Water samples for CT (total dissolved inorganic carbon) and pHT (pH at the total scale) measurements were taken once per week from two random replicates in the Kiel experiment. CT was measured coulometrically (AIRICAâ CT analyser; Marianda, Kiel, Germany), and pHT was determined with a 826-Metrohmâ (Filderstadt, Germany) mobile pH meter equipped with a Metrohmâ 6.0262.100 electrode using Tris/HCl and AMP/HCl seawater buffers (Dickson et al., 2007). Measurements were corrected using standard reference seawater (Dickson et al., 2003). For the Tja¨rno¨ experiment, water samples for pCO2 determination and pHT measurements were taken twice per week in one random replicate. pHT was determined with a WTWâ 330i pH meter equipped with a SenTixâ 81 pH electrode using Tris/HCl and AMP/HCl seawater buffers. Measurements were conducted in fresh samples and in samples equilibrated with custom air/ CO2 mixtures at 977 and 3000 ppm CO2. All other carbonate system parameters were calculated using the CO2SYS program (Pierrot et al., 2006) with dissociation constants (K1 and K2) according to Millero et al. (2006) and KHSO4 dissociation constant after Dickson (1990). Similar measurements were made weekly from freshly taken field samples at the different study sites (see above).

Response variables In both experiments, we monitored mortality and basal diameter every second week and at the end of the experiments. We measured dry weight (DW), ash weight (AW) and shell strength of barnacles and calculated their condition indices (CI). The study in Tja¨rno¨, Sweden was run on limited shortterm grant money during a summer visit and does therefore only comprise a limited number of response variables. For the Kiel experiment, however, we also assessed moulting frequency, reproduction and net-calcification rates in adult barnacles, as well as larval settlement and postsettlement growth of F1 offspring from the adult individuals in the experimental treatments. The surfaces of barnacle shells were also investigated using scanning electron microscopy (SEM). In all cases, mean values were calculated for each aquarium (=replicate).

Mortality, growth, and CI. Barnacle mortality and growth were assessed from digital images of the back of each transparent settlement panels (Canon EOS camera, EFS 18–55 mm). Images were taken every second week, and the maximum basal diameter of each barnacle was determined using image analysis (ImageJ 1.43u). DW, AW, and CI were determined for six haphazardly selected barnacles scraped from each panel (Kiel: week 2, 6, 12, 20; Tja¨rno¨: week 2, 4, 5). These barnacles were frozen at 20 °C, dried at 80 °C for 24 h and weighed (Sartoriusâ, G€ ottingen, Germany, 0.1 mg). Samples were burned at 500 °C (Naberthermâ, Lilienthal, Germany, B150) and the remaining inorganic material (AW) was weighed. Ash-free dry weight (AFDW) was calculated as DW-AW and CI as AFDW/AW (Range et al., 2011; Pansch, Nasrolahi et al., 2013).

768 C . P A N S C H et al. Moulting frequency and reproduction (Kiel only). Moulting frequency and rates of larval release (as a measure of reproduction) were determined by placing filters (90-lm mesh size) at the outflow of five experimental aquaria from each treatment combination. Filters were placed for 12 h on 4 different days during weeks 9–12, and numbers of nauplius larvae and adult exuviae released were counted. Data were standardized as larvae released per hour per adult barnacle and as percent of the overall amount of larvae or exuviae released at each sampling day from all investigated barnacle specimens. Data were pooled over the 4 sampling weeks (P for week >0.2; Underwood, 1997). Net-calcification rates (Kiel only). Settlement panels from six replicates from each treatment combination were cleaned of epibionts at the end of the experiment (week 20) and placed into sealed 2-l plastic aquaria containing 1.7 l filtered (0.2 lm) seawater (salinity: 20.7  0.2). The aquaria were aerated with ambient air or the respective treatment pCO2. Water samples were taken at the beginning and at the end of 36-h incubation. AT was analysed by potentiometric titration (VINDTAâ autoanalyzer, Marianda, Kiel, Germany). Measurements were corrected using DICKSON reference seawater (Dickson et al., 2003). Aquaria containing filtered (0.2 lm) seawater but no panels with barnacles served as controls. Net-calcification rates (G; lmol CaCO3 9 g DW 1 9 h 1) were estimated using the alkalinity anomaly technique with the equation: G = DAT 9 2 1 (Smith & Key, 1975; Gazeau et al., 2007; Langdon et al., 2011). Scanning electron microscopy (SEM, Kiel only). Haphazardly selected barnacles from each treatment combination were removed from settlement panels in week 18, dried at 80 °C for 24 h, and coated with gold-palladium. Images were taken using a Zeissâ, Oberkochen, Germany DSM 940 scanning electron microscope.

Shell strength (Kiel only). The force required to break barnacle shells was determined in six haphazardly selected individuals from each treatment combination in week 12 and 20. We used a TAXT2i texture analyser (Stable Micro Systemsâ, Godalming, UK; 25–1 measuring cell) to push a cylinder of 2 mm diameter onto the rostrum/rostrumlateral-plate (Fig. 5) with a speed of 1.0 mm s 1 (Pansch, Nasrolahi et al., 2013). The pressure required to break the shell was measured (Texture Expert Exceed 2.64; see also McDonald et al., 2009). Settlement and growth of F1 larvae and juveniles (Kiel only). Nauplius larvae were collected on 90-lm filters from six replicates from each treatment combination in week 14. Larval culture methods for this species or the related barnacle A. amphitrite were described earlier in more detail (Thiyagarajan, 2010; Pansch et al., 2012). In short, larvae were cultured at 1 larva ml 1 in filtered (0.2 lm) seawater at the same acidification levels as the parental treatments. Larvae were fed every second day with marine diatoms (C. calcitrans and S. costatum, 1 : 1 mixture, total 2 9 105 cells ml 1; Nasrolahi et al., 2007), irrespective of the food treatment

their parents experienced. Water was exchanged every second day. Measurements of pHNBS and temperature were conducted every second day in three replicates from each treatment combination. Water samples for salinity, CT and pHT analyses were taken once per week in two replicates from each treatment combination. Water analyses were conducted as described above. Nauplius larvae develop through six stages before moulting to a nonfeeding cypris stage (Jones & Crisp, 1954). Settlement success of cypris larvae (‘cyprids’) on the culture vessel walls was determined after 16 days. Subsequent growth of juveniles was determined by digital photography and image analysis as described above. Juvenile growth is shown as percent increase in basal diameter between day 34 and day 50 postlarval incubation.

Field controls. Natural growth of barnacles in the field was assessed by exposing settlement panels in the Kiel Fjord and Tja¨rno¨ archipelago at 1.5 m depth on the first day of the respective laboratory experiment. Sampling and analysis procedures were as described for the laboratory experiments. Newly settled barnacles were regularly removed to allow competition-free growth of the monitored individuals. Temperature (HOBOâ Onset Computer, Bourne, MA, USA) and salinity (CTD-logger; STAR-ODDIâ, Gardabaer, Iceland) were logged continuously in the field and water samples were taken once per week to determine carbonate system variables (methods outlined above).

Statistical analysis For both experiments, a fully crossed two-factorial design, with food (2 levels) and acidification (3 levels) as fixed factors, was analysed using permutation-based multivariate analysis of variance (PERMANOVA + 1.0.2 add-on for PRIMER 6.1.12; Anderson et al., 2008; Clarke & Gorley, 2006). All response variables were analysed by PERMANOVA (Size, CI, DW, and AW were analysed using repeated measures PERMANOVA). We also used PERMANOVA for pairwise post hoc comparisons. All statistics were based on 9999 permutations and Euclidean distance matrices.

Results

Carbonate chemistry fluctuations in the study areas Under the comparable 5-week period (weeks 6–12), barnacles growing in the Kiel Fjord experienced greater pH (DpHNBS = 0.43) but lower salinity (Dsalinity = 5.2) fluctuations than those in the Tja¨rno¨ archipelago (DpHNBS = 0.27, Dsalinity = 14.6), while temperature fluctuations did not differ considerably (Dtemperature Kiel = 5.2 °C, Tja¨rno¨ = 4.7 °C; Fig. 1a–c). Corresponding pCO2 fluctuations were much higher in Kiel (DpCO2 = 910 latm) than in Tja¨rno¨ (DpCO2 = 174 latm) with peak values of 1351 and 520 latm, respectively (Fig. 1d). The Kiel Fjord had calcite saturation states © 2013 John Wiley & Sons Ltd, Global Change Biology, 20, 765–777

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down to ΩCa = 1.02. In contrast, ΩCa in the Tja¨rno¨ archipelago never fell below 1.69. Fluctuations outside this period in the Kiel Fjord were even greater (Table S2; Fig. 1a–d). Experimental treatments were saturated with respect to calcite in all (ΩCa = 1.31–3.78) but the highest pCO2 levels (Table S2).

Mortality, growth, and CI of barnacles Mortality of Kiel barnacles was not significantly affected by either food or elevated pCO2 (Table S3; Fig. 2a). Mortality of Tja¨rno¨ barnacles was more than an order of magnitude greater than mortality of Kiel barnacles and increased by 79% at high pCO2 compared with ambient pCO2 at low-food and by 91% at high pCO2 compared with ambient pCO2 at high food (Table S4; Fig. 2b). In Kiel, food availability significantly affected barnacle size (diameter, DW, AW) and CI, irrespective of pCO2 (Table S3; Fig. 2c and e; Fig. S1a,c). Barnacles were 52% larger (mean diameter over all pCO2 treatments) at high-food compared with low food at the end of the 20-week experiment (Fig. 2c). The CI of Kiel barnacles was significantly higher at high food compared with low food over the 20 weeks (Fig. 2e). Field controls in Kiel showed similar sizes (diameter, DW, and AW) as were obtained in the high-food treatments in the laboratory (Fig. 2c and e; Fig. S1a,c). The CI of field control barnacles from the Kiel Fjord ranged between the CI of barnacles from the high- and low-food treatments in the respective experiment (Fig. 2e). © 2013 John Wiley & Sons Ltd, Global Change Biology, 20, 765–777

In Tja¨rno¨, barnacles were significantly larger at high food compared with low food at the end of the 5-week experiment (by 25%, mean diameter over all pCO2 treatments; Table S4; Fig. 2d). Tja¨rno¨ barnacles at low food were significantly smaller at high pCO2 than those at ambient pCO2 (by 12% and by 18% in week 2 and 4, respectively; Fig. 2d). Tja¨rno¨ barnacles at high food were significantly smaller at high pCO2 than those at moderate and ambient pCO2 (by 15% and by 7% in week 2 and 4, respectively; Fig. 2d). By week 5, all significant pCO2 effects on size of Tja¨rno¨ barnacles had disappeared (Fig. 2d). Barnacles from Tja¨rno¨ were significantly larger (DW and AW) at high food compared with low food from week 2 onwards, irrespective of pCO2 (Table S4; Fig. S1b,d). Tja¨rno¨ barnacles in week 4 had a higher CI at high food, irrespective of pCO2 (Table S4; Fig. 2f). Field controls from the Tja¨rno¨ archipelago showed slightly higher growth as well as higher CIs compared with all treatment combinations in the respective laboratory experiment over the entire experimental period (Fig. 2d and f).

Moulting frequency and reproduction (Kiel only) Moulting frequency in Kiel barnacles was significantly elevated under increasing pCO2, irrespective of food (by 26% and 59% at moderate and high pCO2 compared with ambient pCO2, respectively, means over all food treatments; Table S3; Fig. 3a). Kiel barnacles at high food released significantly more larvae than Kiel

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Fig. 2 Mortality (a, b), Size (c, d) and condition index (e, f) of Amphibalanus improvisus from Kiel (left) and Tja¨rno¨ (right) in the field (indicated by +) and at high and low food and ambient, moderate and high pCO2 (means  95% CIs; N = 8 Kiel, N = 6 Tja¨rno¨; n.a. = not available). Significance of effects (PERMANOVA) is indicated by *P < 0.05, **P < 0.01, ***P < 0.001 (Table S3, S4). Food and acidification treatments differ when they do not share a line connection or a lower case letter, respectively (within weeks; PERMANOVA pairwise tests at P < 0.05).

barnacles at low food, irrespective of pCO2 (by 87%, means over all pCO2 treatments; Table S3; Fig. 3b).

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SEM photographs of Kiel barnacles showed increasing corrosion of the outer shell with increasing pCO2 and slightly with increasing food availability (Fig. 4).

Net-calcification rates of Kiel barnacles were significantly reduced with increasing pCO2, irrespective of food (by 26% and 69% at moderate and high pCO2 compared with ambient pCO2, respectively, means over all food treatments; Table S3; Fig. 3c). Only the low food 9 high pCO2 treatment combination made individuals suffer from net-dissolution but mean net-calcification rates were always positive, irrespective of food or pCO2 (Fig. 3c).

Shell strength (Kiel only) The force required to break the shells of Kiel barnacles was significantly higher at high food compared with low food, irrespective of pCO2 (by 43%, means over all pCO2 treatments and sampling days; Table S3; Fig. 3d). Field controls in Kiel showed a similar mean shell © 2013 John Wiley & Sons Ltd, Global Change Biology, 20, 765–777

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Fig. 3 Moulting frequency (a), reproduction (b), net calcification (c) and shell strength (d) of Amphibalanus improvisus from Kiel in the field (indicated by +) and at high and low food and ambient, moderate and high pCO2 (means  95% CIs; N = 5 (a, b), N = 6 for (c), N = 8 (d); n.a. = not available). Significance of effects (PERMANOVA) is indicated by *P < 0.05, **P < 0.01, ***P < 0.001 (Table S3). Food and acidification treatments differ when they do not share a line connection or a lower case letter, respectively (within weeks; PERMANOVA pairwise tests at P < 0.05).

strength as the high food 9 ambient pCO2 treatment combination in week 12 (Fig. 3d).

Settlement and growth of F1 larvae and juveniles (Kiel only) Settlement of F1 larvae and growth of F1 juveniles of Kiel individuals were not significantly affected by elevated pCO2 or the food treatment of the parents (Table S3; Fig. 5).

Discussion In both populations, food availability had a much stronger impact on the performance of the barnacle Amphibalanus improvisus than elevated pCO2 (Table 1). Enhanced food availability increased the resilience of individuals to CO2 stress. The A. improvisus population from the more environmentally variable Kiel Fjord proved to be less sensitive to acidification treatments than the population from the more stable Tja¨rno¨ archipelago (Table 1). This enhanced tolerance of Kiel barnacles to elevated pCO2 was also carried-over from adults to their offspring (Table 1). © 2013 John Wiley & Sons Ltd, Global Change Biology, 20, 765–777

Many marine species, including the barnacle Semibalanus balanoides, show reduced survival at elevated pCO2 (Findlay et al., 2009, 2010a,b; Findlay, Burrows et al., 2010; Harvey et al., 2013) – a result that was also observed in A. improvisus from Tja¨rno¨ in the present study. In contrast, mortality in A. improvisus individuals from Kiel was unaffected even under severely elevated pCO2 (see also Pansch, Nasrolahi et al., 2013; McDonald et al., 2009 for the closely related barnacle species A. amphitrite). In both populations of A. improvisus, food availability had no impact on mortality but was the main driver for growth. Individuals grew about 50% faster and invested more into body growth relative to shell growth at elevated food levels. Although physiological stress due to elevated pCO2 has the potential to alter growth (Kroeker et al., 2010, 2013; Harvey et al., 2013) and CI (20% decreased CI in Crassostrea gigas at ca. 1500 latm pCO2; Lannig et al., 2010), these traits seemed not to be impacted by highly elevated pCO2 in the Kiel population of A. improvisus. Tja¨rno¨ individuals of A. improvisus, however, were affected even by moderate acidification levels predicted for the open ocean by the end of this century (Caldeira & Wickett, 2005)

772 C . P A N S C H et al. 1

2

high-food

ambient pCO2

moderate pCO2

low-food

2

1

high pCO2

Fig. 4 SEM photographs of Amphibalanus improvisus from Kiel. Grey circles illustrate the point where the force for the shell strength measurements was applied. Black arrows display areas of damaged epicuticula (1) and dissolution of the outer calcite layers (2).

(b) Ambient Moderate High

80 60 40 20 0

Postlarval growth (%)

Larval settlement (%)

(a) 100

100 80 60 40 20 0

Low food

High food

Adult food

Low food

High food

Adult food

Fig. 5 Larval settlement (proportion of larvae settled) and juvenile growth (b) of Amphibalanus improvisus F1 individuals from Kiel at different pCO2 treatments from adults kept at high food and low food and ambient, moderate or high pCO2 [means  95% CIs; N = 6 (a); N = 4 (b)]. PERMANOVA results are given in Table S1).

under reduced food, although they could compensate for this stress when sufficient food was available (Fig. 1). The fact that the impact of elevated pCO2 on

growth of Tja¨rno¨ individuals could not be detected after prolonged treatment was likely due to high selective mortality (sensitive individuals died by week 5) © 2013 John Wiley & Sons Ltd, Global Change Biology, 20, 765–777

H A B I T A T A N D F O O D D E T E R M I N E O A S T R E S S R E S P O N S E 773 Table 1 Summary table of the effects of food and acidification on the barnacle Amphibalanus improvisus (↑ = positive effects, ↓ = negative effects, ○ = no significant effect)

Kiel population Survival Size Dry weight and ash weight Condition index Moulting frequency Reproduction (larval release) Net-calcification Shell strength Shell shape Development of F1 larvae Size of F1 juveniles Tja¨rno¨ population Survival Size Dry weight and ash weight Condition index

Food availability

Elevated pCO2

○ ↑ ↑ ↑ ○ ↑ ○ ↑ ○ ○ ○

○ ○ ○ ○ Increased ○ ↓ ○ ↓ ○ ○

○ ↑ ↑ ↑

↓ ↓ ○ ○

and a reduced sensitivity of the survivors. This is indicative of substantial potential for selective adaptation in this species. As salinity has the potential to impact A. improvisus (Lind et al., 2013 and references therein) and it had been demonstrated that this species thrives well under low salinity conditions (Fyhn, 1976), additive effects of elevated pCO2 and salinity, however, cannot be excluded. Nevertheless, there are many data that show juveniles of this species to be tolerant to salinity shifts (Nasrolahi et al., 2013) at 20 °C (common summer water sea surface temperatures) as used in the present study. Barnacles moult naturally every 2–3 days (Costlow & Bookhout, 1957) and we observed that moulting frequency increased almost linearly with seawater pCO2 in individuals from Kiel. Kurihara et al. (2008) found that moulting frequency in the shrimp Palaemon pacificus increased at pCO2 of ca. 1000 latm and decreased at ca. 1900 latm as a possible consequence of shifts in metabolic activity. In our study, however, moulting frequency did not correlate with altered growth or the CI, and for the moment, we cannot explain this response pattern. Consistent with results for other marine invertebrates (Marshall et al., 2008; Ries et al., 2009; Nienhuis et al., 2010; Melzner et al., 2011; Ries, 2011) we found that netcalcification of A. improvisus from Kiel was strongly reduced under increased pCO2. We also observed severe shell corrosion at high pCO2 (Ωca < 1, corrosive) but corrosion was negligible at moderate pCO2 (Ωca > 1). With the exception of some individuals at the © 2013 John Wiley & Sons Ltd, Global Change Biology, 20, 765–777

highest pCO2 treatment, however, net-calcification was positive. Barnacles produce the more stable form of CaCO3, (97% calcite in shells; Findlay et al., 2010a; Bourget, 1987; Barnes et al., 1976) and an epicuticula covers the outer shell layer (Costlow, 1956) preserving it from direct exposure to undersaturated corrosive water. Furthermore, calcification in crustaceans occurs in compartments isolated from the external environment, where HCO3 is used for CaCO3 precipitation rather than CO32 (Whiteley, 2011). These traits might be responsible for the fact that significant amounts of CaCO3 were built and maintained by barnacles, even under corrosive conditions. Nevertheless, signs of a damaged epicuticula were observed locally (Fig. 5) that might have exposed CaCO3 structures to corrosive waters and enhanced shell dissolution (Ries et al., 2009; Rodolfo-Metalpa et al., 2011). Similar effects were previously observed in the blue mussel Mytilus edulis (Thomsen et al., 2010). Elevated pCO2 did not, however, affect the shell strength of A. improvisus from Kiel. This is consistent with earlier results for this species (Pansch, Nasrolahi et al., 2013). Although elevated pCO2 has been observed to delay embryonic development in the barnacle Semibalanus balanoides (Findlay et al., 2009), we observed no impacts on reproduction (i.e., the amount of larvae released) of A. improvisus from Kiel – a result that is consistent with outcomes for other crustaceans, including the barnacle species, A. amphitrite (Mayor et al., 2007; Kurihara, 2008; McDonald et al., 2009). Elevated food, however, favoured the reproduction of A. improvisus from Kiel (see also Hines, 1978). Early life-history development is considered to be a critical stage in many marine species (Pineda et al., 2012). We found, however, that naupliar development, metamorphosis to cypris, and metamorphosis to the juvenile stage were not impacted by elevated pCO2 in A. improvisus from Kiel. Barnacle cyprids utilize energy reserves gained during previous larval phases (6 naupliar stages) for metamorphosis and early postlarval development (Anderson, 1994; Thiyagarajan et al., 2003). These processes are associated with high mortality (Gosselin & Qian, 1997; Jarrett, 2003; Thiyagarajan et al., 2005; Shanks, 2009; Gosselin & Jones, 2010) and are considered critical in the ontogeny of barnacles (Thiyagarajan et al., 2002; Nasrolahi et al., 2013). Our results on A. improvisus, in contrast, confirm the previously described low sensitivity of early life-history stages of this species (Pansch et al., 2012; Pansch, Schlegel et al., 2013 see also McDonald et al., 2009). Most studies to date neglect the effects of adult experience on offspring performance (‘carry-over effects’, Pechenik et al., 1998). Here, we demonstrated clearly that adult experience (in terms of food and

774 C . P A N S C H et al. acidification) did not affect the development of F1 larvae to juveniles in A. improvisus from Kiel. This again, highlights the tolerance of this population of A. improvisus to elevated pCO2 over the entire life cycle (Pansch et al., 2012; Pansch, Nasrolahi et al., 2013; Pansch, Schlegel et al., 2013). Results obtained from an OA ( 0.8 pH units) stress response study on one population of the closely related barnacles species A. amphitrite, showed that OA did not affect larval condition, cyprid size, cyprid attachment and metamorphosis, juvenile to adult growth and egg production (McDonald et al., 2009). In contrast, these authors observed slight changes in shell hardness and shell composition (McDonald et al., 2009), effects that could not be verified within the present study. This, again, illustrates certain populations of this genus of marine calcifiers to be rather tolerant to future OA conditions over its entire life cycle. In other locations, reduced pCO2 has been reported to have negative effects on barnacles: natural growth rates of the acorn barnacle Balanus glandula were reduced during upwelling events along the central coast of Oregon, USA (Sanford & Menge, 2001; see also Skinner et al., 2007; Phillips, 2005) – a result that is possibly explained by undersaturation of upwelled seawater. More generally, barnacles can be found in shallow, tidal or atidal areas worldwide from fully marine to almost brackish waters (e.g., Fyhn, 1976; Foster, 1987) and are thus naturally exposed to fluctuating and low pH due to river runoff, upwelling or strong biological activity (Duarte et al., 2013). These processes might be even more pronounced in brackish waters due to its lower buffering capacity. During the second half of our 20-week experimental period, upwelling of highpCO2 water into shallow waters within Kiel Fjord created calcite saturation states as low as 1.0, on the threshold of being corrosive to calcite shells of barnacles (Barnes et al., 1976; Bourget, 1987; Findlay et al., 2010a). Nevertheless, calcifiers such as A. improvisus as well as Mytilus edulis are abundant in this habitat and can even dominate benthic hardbottom communities in the Kiel Fjord (D€ urr & Wahl, 2004; Thomsen et al., 2010, 2013). This further supports the findings from the laboratory studies (present study, Pansch, Nasrolahi et al., 2013). It is possible that natural fluctuations in the environment have led the Kiel population of A. improvisus to be preselected to elevated pCO2. Seasonal fluctuations in carbonate chemistry of this shallow coastal bay can be tremendous, which over many generations has created a strong selection pressure (Schmidt & Rand, 2001; Marshall et al., 2010; Sanford & Kelly, 2011) that has increased tolerance of this barnacle population to elevated pCO2. Although equivalent long-term data

sets are not available at this point for the Tja¨rno¨ Archipelago, this study and earlier investigations (Larsson, 2010; J. Havenhand & C. Pansch, unpublished data) show this habitat to be much less variable than the Kiel Fjord with respect to carbonate chemistry. This may explain the higher sensitivity of the Tja¨rno¨ population to elevated pCO2. Comparisons of polar and subpolar populations of the barnacle S. balanoides have also found population-specific differences (Findlay et al., 2009, 2010a,b; Findlay, Burrows et al., 2010). Comparisons of Mytilus spp. from the North Sea and Baltic Sea also indicate that long-term acclimation to elevated pCO2 in the Baltic Sea likely increases the ability to calcify under acidified conditions (Michaelidis et al., 2005; Ries et al., 2009; Thomsen et al., 2010, 2013). As seen for other life stages of A. improvisus (Pansch, Schlegel et al., 2013) as well as for other species (Schlegel et al., 2012), we found strong variability in the responses of individuals to changes in seawater pCO2, with some individuals even doing well under acidification. Given this variability, if ocean acidification develops to exert strong selective pressure, we are likely to see emerging adaptation of the Tja¨rno¨ population to future OA (see also Lohbeck et al., 2012). In summary, A. improvisus from Kiel Fjord seem to tolerate high levels of acidification well (see also Pansch et al., 2012; Pansch, Nasrolahi et al., 2013). Even carryover effects of elevated pCO2 were not observed for this population. Our findings strongly support the hypothesis that populations from fluctuating pCO2 environments are more tolerant to elevated pCO2 than populations from more stable pCO2 habitats. It seems clear, however, that atmospheric CO2 concentrations will continue to rise (IPCC, 2013) and can drastically amplify acidification in coastal waters (>4000 latm pCO2; Melzner et al., 2013). In the course of global change, many parameters will change, and warming as well as eutrophication (resulting in increased food supply; present study), can mitigate the negative impacts of future OA (Pansch et al., 2012; Pansch, Nasrolahi et al., 2013; this study). Consequently, it seems clear that predicted changes in OA for the next two centuries does not represent a major direct threat for the barnacle A. improvisus. OA may nonetheless influence the structures of the communities in which A. improvisus lives, due to species-specific differences in sensitivities to OA within those communities (Fabricius et al., 2011; Hale et al., 2011). Projections from open-ocean models as well as from the responses of open-ocean organisms are not relevant for coastal habitats, and consequently, studies with greater spatio-temporal detail are needed to predict future changes in highly productive coastal ecosystems. © 2013 John Wiley & Sons Ltd, Global Change Biology, 20, 765–777

H A B I T A T A N D F O O D D E T E R M I N E O A S T R E S S R E S P O N S E 775 Acknowledgments We thank Sebastian Fessler, Arne Ko¨rtzinger and Mandy Kierspel for pH, CT and AT measurements, Martin Ogemark for invaluable help in the laboratory and Rolf Schmaljohann for providing SEM facilities. This project was financed by the cluster of excellence ‘The Future Ocean’ (Deutsche Forschungsgesellschaft – DFG; Neglected Bottleneck: D1067/34.1), the BioAcid project (Federal Ministry of Education and Research – BMBF; D10/4.1.2; FKZ 03F0608A), the EU FP7 research infrastructure initiative ASSEMBLE (grant agreement no. 227799) and was partly performed within the Centre for Marine Evolutionary Biology (www.cemeb.science.gu.se) which is supported by a Linnaeus grant from the Swedish Research Councils VR and FORMAS. We thank the Sven Lov
en Centre for Marine Sciences – Tja¨rno¨ for kind hosting and collaborations.

Author contribution CP and IS contributed equally to this work. CP and IS collected the data, CP and IS analysed the data and wrote the manuscript. JH and MW contributed to the development of research questions, experimental concept and to revisions. None of the authors have any conflict of interest regarding Wiley-Blackwell guidelines.

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Supporting Information Additional Supporting Information may be found in the online version of this article: Figure S1. Dry weight (a, b) and ash weight (c, d) of Amphibalanus improvisus from Kiel (left) and from Tja¨rno¨ (right) in the field (indicated by +) and at high and low food and ambient, moderate or high pCO2 (means  95% CIs; N = 8 Kiel, N = 6 Tja¨rno¨; n.a. = not available). Significance of effects (PERMANOVA) is indicated by *P < 0.05, **P < 0.01, ***P < 0.001 (Table S1, S2). Food treatments differ when they do not share a line connection (within weeks; PERMANOVA pairwise tests at P < 0.05). Table S1. Feeding treatments in cells per ml of Chaetoceros calcitrans and Skeletonema costatum (1 : 1 mixture) and in numbers of individuals of brine shrimp nauplii (Artemia salina). Total Carbon supply following Troedsson et al., 2005 (C. calcitrans = 4.6  0.3 pg C cell 1), Granum et al., 2002 (S. costatum = 19  4 pg C cell 1) and Hii et al., 2008 (A. salina nauplii = 0.905 lg C Ind 1). Table S2. Water chemistry parameters for field and laboratory experiments (Kiel: June - October 2011, Tja¨rno¨: August–September 2011; data are means  SD). Table S3. Effects of food and acidification over time on the response variables investigated in Amphibalanus improvisus from Kiel (p (MC) = p value after Monte Carlo correction). Statistically significant effects are in bold. Table S4. Effects of food and acidification over time on the response variables investigated in Amphibalanus improvisus from Tja¨rno¨ (p (MC) = p value after Monte Carlo correction). Statistically significant effects are in bold. Data S1. References.

© 2013 John Wiley & Sons Ltd, Global Change Biology, 20, 765–777