Adult acclimation to combined temperature and pH stressors significantly enhances reproductive outcomes compared to short-term exposures

less well. After 17 months, performance was not signiﬁcantly different across treatments, including controls. However, under the altered conditions urchins produced larger eggs compared with control animals. 5. These data show that under long-term culture adult S. neumayeri appear to acclimate their metabolic and reproductive physiology to the combined stressors of altered pH and increased temperature, with relatively little measureable effect. They also emphasize the importance of long-term studies in evaluating effects of altered pH, particularly in slow developing marine species with long gonad maturation times, as the effects of altered conditions cannot be accu-rately evaluated unless gonads have fully matured under the new conditions. variability remained after transforming data following Sokal & Ro-hlf (1995). Proportional data were arcsin transformed prior to analysis (Kelly et al. 2000). Adult growth was assessed for normality (Anderson – Darling), and relationships with time were determined using correlation and regression analyses. Multiple comparisons of treatment slopes were assessed with a GLM of interacting treatments with time and using time as a covariant. Results were accepted after homogeneity of residuals was met. Growth and standard error of slopes were quantiﬁed from the GLM’s resulting coefﬁcient terms. Due to multiple testing, P -values were adjusted for larval morphometrics, development stages and abnormalities using the method of Benjamini & Hochberg (1995). Data are available via the Polar Data Centre (Suckling et al. 2014).


Introduction
To provide more accurate predictions of the consequences of climate change on biodiversity, it is essential to understand the complex synergistic effects of multiple environmental stressors across the different life-history stages of species, and the resulting ecosystem interactions (Crain, Kroeker & Halpern 2008;Kroeker et al. 2010;Kroeker et al. 2011;Russell et al. 2012). Despite considerable research in this area, largely fuelled by concern about the increasing acidification of the World's oceans, there are still critical gaps in our knowledge, such as species' abilities for long-term acclimation or adaptation to altered conditions and their phenotypic plasticity (Munday et al. 2009;Sunday et al. 2014), as most experiments to date have been short-term. Data arising from recent long-term exposure (in excess of a year) and transgenerational studies are revealing contrasting results to the short-term data, with adult acclimation to altered conditions an important factor Miller et al. 2012;Form & Riebesell 2012;Dupont et al. 2013;Calosi et al. 2013). Extended studies are therefore imperative for accurate future predictions.
High-latitude seas are regarded as particularly vulnerable to climate change effects, with regions of the Southern Ocean experiencing rapid rates of warming (IPCC 2013) and predictions that seasonal winter carbonate ion undersaturation will begin by 2030 (McNeil & Matear 2008). In tandem, there is currently very limited knowledge on capacities of this region's fauna to acclimate to altered conditions (Peck et al. 2014). A keystone Southern Ocean species is the sea urchin Sterechinus neumayeri. It is a circum-polar endemic, and in shallow water, it can be the most abundant macrobenthic invertebrate, playing a crucial role in the benthic ecosystem (Brey & Gutt 1991). This species is a major transformer of energy from detrital and depositing sources. It modifies seabed communities by intense grazing pressure, especially at the high densities that S. neumayeri naturally occurs at, limiting the density and diversity of encrusting and attached forms, including macroalage (Bowden et al. 2006;Campana et al. 2009). As a marine calcifier, it also contributes significantly towards global CaCO 3 sequestration within a depth range of 0-800 m (Lebrato et al. 2010;Sewell & Hofmann 2011). Therefore, any negative impacts from climate change on this species will have major impacts on the benthic food web, and also potentially impact global CaCO 3 cycling (Sewell & Hofmann 2011). In common with many Antarctic marine invertebrates, S. neumayeri is stenothermal, with low metabolic rates, slow annual growth and extended longevity, living up to 40 years and requiring up to 6 months to acclimate to elevated temperatures (Brey et al. 1995;Brockington 2001;Peck et al. 2009Peck et al. , 2014Peck, Morley & Clark 2010). Consequently, adaptation to new environmental conditions will be strongly dependant on phenotypic plasticity and acclimatization (Somero 2010).
To date, all acidification studies on S. neumayeri have been short-term, and mostly focussed on pH (Clark, Lamare & Barker 2009;Ericson et al. 2010Ericson et al. , 2012Byrne et al. 2013;Yu et al. 2013), with only two studies on the combined effects of pH and temperature Byrne et al. 2013). Furthermore, all were restricted to early developmental life stages and did not cover the full course of larval development. In each case, the offspring were produced from parents held under ambient pH conditions with the offspring directly introduced into sea water at low pH, which may produce misleading responses for long-term predictions (Suckling et al. 2014a, b). Only temperate and tropical examples exist for adult pre-conditioning of echinoderms prior to spawning over variable time-scales, from 6 weeks in Echinometra mathaei (Uthicke et al. 2013) (Dupont et al. 2013). In the latter example, spawning after 4 months and 16 months (encompassing part and full gametogenesis, respectively) showed marked differences. There was improved fecundity and larval survival at the longer acclimation period, albeit with potentially deleterious carry over effects reported when the larvae and juveniles were cultured under the altered conditions. In S. neumayeri, vitellogenesis takes between 18 and 24 months and spermatogenesis 12 months. Female gonads contain two cohorts of eggs; one in its second year maturing until spawned with another cohort <12 months old and in an early vitellogenic state (Pearse & Cameron 1991;Brockington, Peck & Tyler 2007). In this study, we determined the physiological impacts of altered sea water pH and temperature on adult S. neumayeri over a 2 year period to encompass the full development of both egg cohorts. We aimed to determine whether adult urchins had the physiologically flexibility to cope with the altered culture conditions and to identify the effects of short and long-term adult exposures on the performance of the early life stages of the next generation.

animal collection and culturing
Sterechinus neumayeri were collected by scuba divers in the austral summer of 2008-2009 from 3-10 m depth at Back Bay Lagoon, Ryder Bay, Antarctic Peninsula (67°34 0 S, 68°08 0 W). Specimens were transported to the UK and held in the British Antarctic Survey recirculating aquarium at 0°C (AE0Á1°C) in Cambridge for approximately 2 months before being introduced to the recirculating CO 2 microcosm system [adapted from Widdicombe & Needham (2007) and fully described in Suckling et al. (2014b)]. In the microcosm (200 L), UV sterilized and 50 lm filtered sea water was delivered to 80-L closed cylindrical mixing tanks. CO 2 gas (BOC) was introduced via a ceramic diffuser using an Aquamedic pH controlled computer and electrode system and mixed with sea water by an Aquamedic ocean runner power head 2000. Treated sea water was gravity fed to each experimental tank at a rate of 0Á56 AE 0Á03 L min À1 . The control mesocosm had no pH control system, but did include the Aquamedic ocean runner power head 2000. Sea water quality was assessed every 2-3 days using Nutrafin Aquarium test kits. Ammonia, nitrite and nitrates were maintained well below 0Á4, 0Á2 and 5 mg L À1 , respectively, by a combination of biological filtration, protein skimming and partial sea water exchanges (approximately 5-15% every 2-3 days) to prevent toxicity from metabolic by-products.
Sea water temperature in the experimental microcosms was approximately +2°C (Table 1), with pH initially set at ambient levels in all tanks. Urchins were held in these conditions for 14 days before gradually decreasing the pH in selected tanks in equal, twice daily increments, over a period of 3 days until the desired pH was achieved. Adult urchins (total n = 288; 26Á18 AE 0Á21 mm mean test diameter) were selected at random and distributed between 36 three litre aquaria (n = 8 urchins per aquarium = 164 cm 2 urchin À1 ). In addition, large animals were designated broodstock (31Á18 AE 0Á37 mm mean test diameter; total n = 72) and kept separately within the microcosm (Fig. S1, Supporting information). All animals were fed Pollachius virens at approximately 2% mean body mass every 14 days.
Treatments used in this study were based on the IPCC 'business-as-usual' scenario with the forecasted reduction of 0Á3-0Á5 pH units in oceanic surface waters by 2100 (IPCC 2013). The treatments used for the adult physiology were control at present day conditions,~pH 8Á0; moderate acidification with a reduction of 0Á3 pH units at~pH 7Á7 and high acidification with a reduction of 0Á5 pH units at~pH 7Á5, with all water temperatures elevated to values between 1Á8 and 2Á4°C ('high temperature'; Table 1). A low-temperature control was not used for the adult physiology assessments because the long-term effects of 2°C warming are already known for adults of this species (Peck et al. 2014).

water chemistry
Temperature was recorded daily for all treatments (Digital Testo 106, Alton, UK). Salinity (Tropical Marine Centre V2 Handheld refractometer, Chorleywood, UK), pH NIST (temperature compensated; HANNA bench top meter pH/ORP 115 v pH21-01) and TCO 2 (mmol L À1 ; Ciba Corning TCO 2 Analyzer 965, Olympic Analytical, Malvern, UK) were recorded weekly ( Table 1). The TCO 2 analyzer was calibrated with 2 g L À1 CO 2 standard prior to measurements. Aquamedic pH probes were calibrated with NIST certified pH buffer solutions at least two times per week. Sea water samples were analysed for phosphate and silicate levels following Nickell et al. (2003).

adult metabolic measurements: oxygen consumption
Within each treatment, the animals were divided into two groups. One group (24 adults) was used to measure oxygen consumption across the 2 year experimental period. Oxygen consumption was measured every 4 months for 10 animals randomly selected from this group of 24 in each of the four treatments using closed bottle techniques (Oberm€ uller et al. 2010). In summary, urchins were placed individually into open chambers, without aerial exposure, and left overnight to adjust to conditions prior to closing chambers. Chamber habituation and respiratory measurements were carried out at approximately the same time of day on all measurement days to minimize circadian effects. Oxygen consumption was measured using a Fibox-3 fibre optic oxygen sensor (PreSens Precision Sensing GmbH, Regensburg, Germany) until there was a 15-25% drop in oxygen saturation. Saturation was not allowed to drop below 70% to minimize stress effects (Peck & Prothero-Thomas 2002). Control chambers (without urchins) were run simultaneously, and all calculated rates of oxygen consumption were corrected for controls. Chamber volumes were corrected for animal volumes prior to calculating oxygen consumed. Animal volume, whole animal wet mass (g AE 0Á01) and test diameter (mm AE 0Á1) were recorded for each urchin. The second group was allocated for destructive sampling to obtain ash free dry masses (AFDM) for each treatment at each sampling time point (n = 8). Within each treatment, live wet volumes vs. urchin diameter regressions were used to relate oxygen consumption measurements to animal AFDM.
adult data collectiongrowth, calcification and gonad index Animal volume, whole animal wet and dry mass (AE0Á01 g), test diameter and thickness (measured at mid-test height from interambulacral plates) (AE0Á01 mm), gonad wet and dry tissue mass (AE0Á01 g), AFDM (AE0Á01 g), sex and gonad index were recorded for each animal used in the destructive sampling analyses. Sex was determined from wet-squash preparations of fresh gonad tissue in sea water using phase-contrast light microscopy (Kelly 2001). Gonad index (GI%) was calculated as gonad dry mass/ whole animal dry mass. Gonad and remaining tissues plus tests were oven dried at 60°C to constant mass. Skeleton mass was measured as ash remaining after ignition in a muffle furnace at 470°C for 24 h. For the broodstock, maximum test diameters were recorded before spawning.

spawning and larval rearing
Broodstock urchins were spawned after they had been in the microcosms for 6 months and 17 months. A low-temperature control (0°C; Table S1, Supporting information) was included because S. neumayeri development is hypothesized to have an optimal temperature window of +0Á2 to +1Á7°C (Stanwell-Smith & Peck 1998), just under our proposed elevated temperature treatments, and the effects on larval from adults pre-exposed to elevated temperatures are currently unknown. Low temperature adults were reared under similar husbandry conditions but held instead in the British Antarctic Survey recirculating aquarium at 0°C (AE0Á1°C). Six females and two males were used in each Table 1. Mean (AESE) water parameters in the adult Sterechinus neumayeri microcosm over the course of the experiment following the format of Barry et al. (2010). Values for pCO 2 , Ω calcite, Ω aragonite and total alkalinity were modelled from CO2SYS (Lewis & Wallace 1998) with refitted constants (Mehrbach et al. 1973;Dickson & Millero 1987). Values represent sea water values before and after sea water exchanges Sea water parameter Low-temperature control* High-temperature control À0Á3 pH À0Á5 pH 1609 AE 14 1871 AE 12 1838 AE 11 1877 AE 11 *Calculated data from Cross, Peck & Harper (2015). Sea water temperature and pH NIST values for each experimental year have been presented in Fig. S7 (Supporting information).
spawning trial following Suckling et al. (2014a,b), with three replicates per treatment. In summary, S. neumayeri were spawned using intracoelomic injections of 1-2 mL of 0Á5 M KCl through the peristomal membrane. After injection, urchins were stored iñ 300-mL glass jars filled with 5 lm filtered treatment sea water until gametes were shed. Eggs were distributed across three replicate treatment buckets until they formed a monolayer at the bottom of each bucket. Buckets were sealed (with lids) and contained 12 L of filtered treatment sea water. Sperm from two males were combined, diluted and added, 1-2 mL per bucket following . Sperm concentration was not calculated but was subject to the same dilution series from concentrated sperm across all treatments (1 : 5; Kelly et al. 2000;Suckling et al. 2014a,b). Cultures were gently mixed to encourage fertilization. Spawning, fertilization and larval rearing were all carried out at 12L : 12D and approximately À1°C (air temperature), irrespective of the parental culture temperature, as it was not technically possible to maintain appropriate numbers of replicates for all treatments at all temperatures. Complete sea water exchanges were carried out every 4 days, with larvae retained in a 50-lm mesh sieve in a water bath while buckets were cleaned and filled. Once the stomach had fully formed (~day 21), larvae were fed Dunaliella tertiolecta (Culture Collection of Algae and Protozoa, code 19/6B) at 1500 cells mL À1 every 2nd day. Sea water pH in development trials was maintained at parental culture levels using specialized mixed gases (1000 and 1500 ppm CO 2 blended with air, BOC) or air bubbled through the culture medium (Table S1, Supporting information). Sea water chemistry measurements followed the regime as described above but with twice weekly measurements and daily pH and temperature readings for all cultures. Twenty-four hours after fertilization hatched blastulae densities were quantified for each treatment (three pseudoreplicates) using a 1 mL Sedgewick rafter cell and compound microscope. Larval survival was assessed every 2-5 days in triplicate (per treatment pseudoreplicate, n = 3). Cultures were gently mixed prior to each 15 mL of culture being removed, and all larvae counted under a dissection microscope and then returned to the respective culture. The mean of these pseudoreplicates was then used to represent each treatment replicate. Embryos and larvae were fixed for morphometric measures after 7-8 and 25 days, following McEdward & Herrera (1999) and Kelly et al. (2000). Larval rearing was terminated at 25 days. Offspring were photographed on an inverted compound microscope with digital camera and graticules at relevant magnifications. Calibrations and measurements were then quantified using ImageJ Analysis software, NIH, Bethesda, MD, USA (Abramoff, Magelhaes & Ram 2004). Approximately, 30 larvae per replicate in each treatment were measured for various parameters including post-oral arm length in the most advanced larvae, which was used as a metric of skeletal development.

statistical analyses and data accessibility
Each treatment (temperature and pH) was analysed as an independent factor nesting tank or replicate for oxygen uptake, test diameter, whole animal wet mass (balanced from random removal of unbalanced data), egg size and larval post-oral arm data. Data were analysed in Minitab (Statistical Software version 15, Coventry, UK) using either Nested ANOVA via a general linear model (GLM) (larval data) or one-way ANOVA (adult data) after assessing normality using Anderson-Darling (P > 0Á05) and homogeneity of variance using Levene's test. After significant ANOVA results, Tukey's or Bonferroni's pairwise comparisons were used to determine treatment differences. Nonparametric Kruskal-Wallis tests were carried out where data consisted of ranks or heterogeneous residual variability remained after transforming data following Sokal & Rohlf (1995). Proportional data were arcsin transformed prior to analysis (Kelly et al. 2000). Adult growth was assessed for normality (Anderson-Darling), and relationships with time were determined using correlation and regression analyses. Multiple comparisons of treatment slopes were assessed with a GLM of interacting treatments with time and using time as a covariant. Results were accepted after homogeneity of residuals was met. Growth and standard error of slopes were quantified from the GLM's resulting coefficient terms. Due to multiple testing, P-values were adjusted for larval morphometrics, development stages and abnormalities using the method of Benjamini & Hochberg (1995). Data are available via the Polar Data Centre (Suckling et al. 2014).

Results
Over the 2 years of the experiment, there were no adult mortalities and sea water parameters were stable (Fig. S2, Supporting information). Saturation states of the microcosm control tanks were slightly undersaturated with respect to aragonite (Ω < 1), but calcite was supersaturated (Ω > 1). Saturation states for lowered pH sea water (À0Á3 and À0Á5 pH) were undersaturated with respect to calcite and aragonite (Table 1), which was probably due to the cold temperatures (Guinotte & Fabry 2008). Sea water chemistry parameters for the larval experiment were very similar to those of the long-term adult treatments (Table S1, Supporting information).

Somatic growth
There was no somatic growth in the adults, as test diameters; whole animal wet and dry mass ad AFDM showed no significant change over the 2 year experiment (Figs S3-6, Supporting information).
developmental stage and abnormal development The cultures were evaluated both for developmental stage and morphometric measurements with data presented here for the final 25 day time point only.

17-month trial
At 25 days, these larvae were more highly developed than those in the 6-month trial, with higher proportions at the later prismatic and pluteus stages in each culture (Fig. 5b). Larvae from low-temperature controls (0°C) were generally the least advanced with the highest proportion of gastrulae when compared with other treatments (F (3,8) = 11Á17, P = 0Á007). The high-temperature controls (+1Á8°C) were the most advanced (Fig. 5b). The proportion of abnormal development was 19% significantly higher under lowest pH conditions (À0Á5 pH) compared with both the low-and high-temperature controls (F (3,8) = 6Á43, P = 0Á029; Fig. 5b). At 17 months, there were pluteus stage larvae with measureable post-oral arms in all cultures, which were not significantly different in length at approximately 53 lm in all treatments (treatment: F (3,54) = 0Á42, P = 0Á741; replicate: F (4,54) = 4Á47, P = 0Á003).

Discussion
adult performance under long-term culture Adults were kept for 2 years in these experimental conditions, without mortality or any apparent effects on growth, feeding, behaviour. There was no significant change in size or mass of the urchins during the 2-year culture (Figs S3-7, Supporting information). This result correlates with previous calcein marking mark-recapture experiments, which showed that S. neumayeri grows very slowly, particularly in the large size range used here, with test diameters above 20 mm (Brey et al. 1995;Brockington 2001). Importantly, the tests did not lose mass throughout the 2 year exposure, which suggests that there was no effect on calcification despite aragonite and calcite being undersaturated, which means that the animals were able to protect the test from dissolution.
Over the 2-year period, oxygen consumption showed a very clear pattern. Short exposure periods (0 month) had significantly higher values than the rest of the experimental period, with urchins in the lowest pH having the highest  Fig. 3. Egg sizes of S. neumayeri exposed to low-temperature (■) and high-temperature ( ) controls and reduced pH conditions À0Á3 pH ( ) and À0Á5 pH (h) after 6 and 17 months.
respiration rates (Fig. 1). It is likely that the decrease in oxygen consumption after month 8 was due to the successful acclimation to the new conditions. The metabolic rates of the animals investigated here exhibited the classic biphasic response to increase in temperature (Bouchard & Guderley 2003). There was an initial 'stress' response as the animals' physiology responded to the increase in temperature, which was followed by a gradual lowering of metabolism to a new steady state as the animals adjusted their physiology to the new conditions, demonstrating physiological flexibility with respect to the altered environments (Piersma & Drent 2003). A similar pattern was shown in resting metabolic rates in the Antarctic fish Pagothenia borchgrevinki when acclimated to 5°C, albeit at much faster rates (Robinson & Davison 2008). However, our experiments included exposure to low pH and it is likely that physiological processes were also adjusting to the undersaturated carbonate conditions. Diffusion of CO 2 into internal body fluids (hypercapnia) leading to a reduction in internal pH (acidosis) has been reported to affect physiological processes (e.g. metabolism; P€ ortner 2008; Melzner et al. 2009;Collard et al. 2013). Homeostasis of inner fluids incurs enhanced costs and elevated respiration plus the diversion of energy away from other processes such as reproduction (P€ ortner, Bock & Reipschl€ ager 2000;Melzner et al. 2009;Collard et al. 2013). Reports for S. droebachiensis showed a negative impact on acid-base homeostasis after 5 days (Spicer et al. 2011), which disappeared after 10-45 days exposure (Stumpp et al. 2012). The rates of oxygen consumption after 8 months were still higher than the average data reported for S. neumayeri in the field (Brockington & Peck 2001), but the animals in our experiment were kept at around 2°C and oxygen consumption rates here were similar to those reported from measurements conducted during the austral summer when temperatures usually increase above 0°C ( Fig. 4. Survival of S. neumayeri offspring (%; AESE) derived from adults exposed for (a) 6 months and (b) 17 months to control and forecasted pH conditions. = Lowtemperature control, = high-temperature control, = À0Á3 pH and = À0Á5 pH.
Thermal acclimation trials previously conducted on S. neumayeri showed that it takes up to 6 months to acclimate to 3°C (Peck et al. 2014). The animals in this experiment may have taken slightly longer to acclimate to the altered conditions (circa 8 months from the oxygen consumption data) due to the interacting effects of temperature and pH (c.f Clark et al. 2013), especially since oxygen consumption levels in high-temperature controls were always lower than those of urchins in lowered pH. Metabolic rates have been measured in other invertebrate ocean acidification studies, with mixed results. Increased metabolic rates were observed in the urchin Paracentrotus lividus, the brittle star Amphiura filiformis and selectively bred oysters Saccostrea glomerata (Wood, Spicer & Widdicombe 2008;Parker et al. 2011;Catarino, Bauwens & Dubois 2012). However, no increase in metabolic rate was observed in wild populations of S. glomerata or the temperate urchin, S. droebachiensis when subjected to altered pH (Parker et al. 2011;Stumpp et al. 2011). These conflicting data may be species/population dependent, but the time of exposure to altered conditions might also be important; sufficient time may not have elapsed for acclimation processes to be completed, as was demonstrated here and the 16-month study of Dupont et al. (2013). In the latter, metabolic rates were not measured, but the lack of impact on fecundity after 16 months of culture, suggested that the urchins were able to replenish energy supplies for successful reproduction, which was taken to indicate full acclimation (Dupont et al. 2013).
Overall, there appeared to be no major long-term effects of altered conditions on adult S. neumayeri beyond 8 months exposure. The lack of major effects may be due to inherent variability in the natural pH of the coastal Southern Ocean. Significant seasonal variation in pH has been recorded in two coastal sites (Prydz Bay and the Ross Sea) with threefold changes in the aragonite saturation state (McNeil, Sweeney & Gibson 2011) across the year equivalent to 0Á3-0Á5 pH units . Thus, shallow coastal populations of S. neumayeri may already be habituated to variable sea water pH.

larval performance under long-term culture
The spawning and larval culture trials conducted after 17 months showed enhanced survival and larval development than the 6-month trial. In these experiments, it was possible to include a low-temperature control (0°C) spawning at current pH, in addition to the high-temperature control (+1Á8°C), which was a useful extra comparison, as technical constraints required all larvae to be reared at the same temperature. At 6 months, the eggs produced by the low-temperature control (0°C) females were larger compared with the other treatments, with a higher rate of hatching success (63%) (Fig. 3). However, after 17 months exposure hatching success (36-47%) and larval survival were similar across all treatments. This agrees with Dupont et al. (2013), where fecundity and larval survival 'recovered' after increased adult exposure, which was attributed to acclimation in S. droebachiensis. In this study, the spawned animals under lowest pH conditions produced larger eggs after extended adult exposure time. This potentially indicates that acclimation has resulted in a higher investment per egg, as a mechanism to increase developmental success and recruitment (Llodra 2002). Larger eggs produce better targets for sperm, thus increasing fertilization success (Levitan 1993). Egg size also correlates with juvenile performance (Emlet & Hoegh-Guldberg 1997).
Whilst survival of embryos and larvae improved with adult exposure time to altered sea water pH, there was also a parallel increase in abnormal larval development for larvae raised from animals kept in high temperatures. These adults were all cultured at around 2°C and abnor- of 25 day old S. neumayeri offspring derived from adults exposed for (a) 6 months and (b) 17 months to low-and high-temperature controls and forecasted pH conditions. B = blastula, G = gastrula, Pr = prismatic larva, Pl = pluteus and A = abnormal development.
mal development in their progeny doubled after 17 months while abnormalities in low-temperature controls (0°C) remained at similar levels throughout the 2 years (Fig. 5). Temperature was previously shown to significantly affect S. neumayeri larval development, with an optimal temperature window of +0Á2 to +1Á7°C proposed for embryo viability (Stanwell-Smith & Peck 1998;Tyler, Young & Clarke 2000). Therefore, it is likely that levels of abnormal development observed here were due to a cumulative temperature effect. However, a treatment effect was also observed, with most abnormal larvae produced in the lowest pH conditions (À0Á5 pH) (Fig. 5). This potentially indicates an interacting effect of exposure to two different stressors. In a previous study, settlement success and metamorphosis was the same across control and reduced pH treatments in the temperate urchin Psammechinus miliaris and less competent larvae appeared to fail earlier under reduced pH compared to control conditions (Suckling et al. 2014b). The increase here in abnormally developed larvae after 17 months adult preexposure, may again be a result of adult acclimation effects, with less competent larvae surviving longer under reduced pH conditions, but this can only be verified by using longer culture periods. Actual numbers of offspring surviving to 18 days postfertilization did show differences between treatments. At the 6-month spawning, the numbers of surviving larvae derived from the parents maintained in reduced pH were significantly lower compared with the other treatments. Conversely, at the 17-month spawning, survival was lowest for larvae derived from the low-temperature control (0°C) and lowest pH (À0Á5 pH) cultured parents, indicating there was little difference between treatments. In each spawning, the larvae were cultured for 25 days. In a temperate species, we previously demonstrated the importance of culturing larvae until settlement (Suckling et al. 2014b), but the slow development of the Antarctic S. neumayeri larvae precluded this, as at least 115 days are required to reach metamorphosis (Bosch et al. 1987). Predicting the final effect of the culture conditions on recruitment and juvenile development can be complex, as demonstrated by Dupont et al. (2013). When they analysed juvenile survival from 4-month acclimated adults, they identified a potential carry over effect with reduced juvenile survival 3 months post-settlement in some treatments. However, these results may have been confounded by high mortality rates a result of potential technical problems in their system, which averaged 33-54% with 95% in juveniles raised at high pCO 2 .
The beneficial effects of allowing adults to acclimate to altered sea water pH, prior to spawning, as demonstrated here, have also been reported for marine invertebrates from lower latitudes (Parker et al. 2011;Dupont et al. 2013;Suckling et al. (2014b). Studies which included the maturation of gonads under altered conditions are rare and show variable results (Parker et al. 2011;Uthicke et al. 2013) and focussed largely on offspring success (Dupont et al. 2013). The experimental time-frame is critical (Suckling et al. 2014b). Even if adults acclimate quickly, gametogenic cycles can take much longer to complete, for example the tropical sea urchin, E. mathaei has a gametogenic cycle encompassing 5-6 months (Pearse 1969), which is much longer than the timing of adult metabolic acclimation (Uthicke et al. 2013).
The fact that embryos and larvae were able to develop in lowered pH with elevated temperatures agrees with previous studies on S. neumayeri. Ericson et al. (2012) observed reduced frequencies of normally developing cleaving embryos at pH 7Á5 with an 11% reduction in both fertilization success and normally developed blastulae. Whilst Byrne et al. (2013) showed that larval growth was reduced at lower pH, but that size was partially compensated for by temperature. They also observed more abnormal larvae and smaller echinoplutei with decreased arm lengths at low pH and elevated temperature. These previous studies are not directly comparable with our data as they used offspring derived from adults held in ambient conditions and without significant gametogenesis under the altered conditions. However, our data confirm these previous findings with the demonstration of a significant temperature effect, further emphasizing the requirement to include both temperature and pH in marine acidification experiments.
One further aspect relevant to these studies is that of source populations and population genetic variability. All of the experiments listed to date on S. neumayeri (with the exception of this study) were performed with Ross Sea populations. Populations of circumpolar species from the Antarctic Peninsula differ significantly in their physiology compared with the same species from the Ross Sea. Acclimation capacities of notothenioid fish (Bilyk & deVries 2011) or the thermal tolerances of marine invertebrates (Morley et al. 2012) differ, even though the ambient temperature regimes differ by only 1-2°C. The basis for this remains unknown, but there is probably a genetic component. Such an effect was demonstrated in an F1 selection experiment on Strongylcentrotus purpuratus urchin populations from along a 1200 km stretch of the United States coastline. Offspring were raised under a variety of altered pH conditions and showed allelic selection on genes for several biochemical functions, including membrane composition and ion transport (Pespeni et al. 2012). In the Southern Ocean, the main area with significant warming is the western Antarctic Peninsula and this is where biodiversity is potentially most at threat. Hence, in these populations, there is a need to understand not only how much phenotypic plasticity exists, but also the fine detail of circumpolar population structure and the consequences for functional biodiversity. These data are needed in order to better predict the effects of climate change on Antarctic marine species (Somero 2010;Peck 2011).

Conclusions
Sterechinus neumayeri adults successfully acclimate in long-term culture to the combined stressors of reduced pH and elevated temperature, with relatively little measurable effect on metabolism and reproduction after 2 years. Since these animals were exposed to altered conditions at rates much faster than is occurring naturally, they seem highly resilient to such changes. At the end of 2 years the animals under lowest pH conditions (À0Á5 pH) were producing fewer, but larger eggs. But losses during development and metamorphosis are high and selection may operate at different rates under the different treatments. Thus, it is only with culture through to settlement and raising of juveniles that accurate predictions can be made for future recruitment rates. These data highlight the importance of experiments using long-term culture in altered conditions for marine species, especially those with poor acclimation abilities. Data here also emphasize that sampling following rapid introduction to reduced sea water pH conditions (e.g. up to 8 months for polar species and likely many weeks for temperate taxa) may be evaluating animals in physiological transition and not acclimated. Exposing adults long term, so that the full gametogenic cycle is undertaken in altered conditions, significantly affects reproductive success and larval outcomes and therefore this practise should be implemented in future studies to increase the accuracy of predictions with regard to species' response to climate change.

Supporting Information
Additional Supporting Information may be found in the online version of this article. Table S1. Mean (AESE) seawater parameters of the Sterechinus neumayeri larval cultures derived from parents pre-exposed to low temperature and high temperature seawater controls and lowered carbonate conditions. Fig. S1. Experimental set-up for each of the treatments with two tray tanks for each treatment, fed by a single mixer tank.    S4. Mean whole animal wet mass (g; AESE) of adult S. neumayeri exposed to control and reduced pH conditions. Fig. S5. Mean whole animal dry mass (g; AESE) of adult S. neumayeri exposed to control and reduced pH conditions. Fig. S6. Mean whole animal ash free dry mass (g; AESE) of adult S. neumayeri exposed to control and reduced pH conditions. Fig. S7. Skeleton mass (measured as ash remaining after ignition) (g; AESE) of adult S. neumayeri exposed to control and reduced pH conditions.