Introduction

Burning of fossil fuels has increased CO2 concentrations in the atmosphere by nearly 70 % compared to pre-industrial levels. Increased carbon dioxide is predicted to increase global average temperature by 1.5–4.5 °C towards the end of this century (depending on representative concentration pathways, RCPs; Meinshausen et al. 2011). Average sea water temperatures are predicted to follow this trend with best estimates for global sea surface temperature (SST) increase in the range of 1°–3° (Collins et al. 2013). Although extensive variation of SST increase is expected for different geographic regions (Brierley and Kingsford 2009), estimates for the Great Barrier Reef are well within this range (Hobday and Lough 2011). Over 30 % of the CO2 emitted is absorbed by the world’s ocean (Sabine et al. 2004). The increased partial pressure of CO2 leads to higher dissolved inorganic carbon, low calcium saturation states, and reduced ocean pH (i.e., ocean acidification, OA). Depending on models and measures taken to reduce carbon emissions, pCO2, values between 420 and >1,370 are expected by the end of this century (Moss et al. 2010; Meinshausen et al. 2011). Ocean pH has already been reduced by 0.1 units compared to pre-industrial levels and is expected to further decrease by 0.3–0.5 units towards the end of this century (Caldeira and Wickett 2005). Thus, marine flora and fauna face a twofold challenge in the near future needing to adapt to higher temperatures and decreased pH/increased pCO2.

Both elevated temperatures and low pH/hypercapnia can be stressors for marine invertebrate developmental stages, juveniles, and adults (Nguyen et al. 2009; Ries et al. 2009; Byrne 2011, 2012; Byrne and Przeslawski 2013).

Increased temperature may be stressful for marine invertebrates, especially tropical species many of which already live at the upper thermal limit of marine isotherms (Hoegh-Guldberg 1999). Ocean acidification is considered to be particularly deleterious for calcifying invertebrates, because calcification may be impaired by the associated reduction in carbonate mineral saturation (Doney et al. 2009). However, as pH, pCO2, and mineral saturation are co-varying stressors, it is difficult to understand which of these are most important (Byrne et al. 2013). For echinoderms, which transport bicarbonate (not carbonate) at the site of calcification and use respiratory CO2 as a source of inorganic carbon (Sikes et al. 1981; Stumpp et al. 2012a), hypercapnia (pCO2), not mineral saturation, appears to be the most important stressor. In addition, a recent synthesis of data on the response of calcification in echinoplutei across world climatic regions to ocean acidification conditions also indicated that hypercapnia was the more important stressor, although tropical species were more sensitive to decreased mineral saturation (Byrne et al. 2013). Recent transcriptomic analyses of echinoplutei reared under ocean acidification conditions indicate a change in biomineralisation, calcium and ion transport, and metabolic genes (Evans et al. 2013).

Although ocean warming and acidification will most likely occur in combination unless carbon emissions are drastically reduced, the interactive effects of these to stressors are poorly understood (Byrne and Przeslawski 2013). In tropical corals, calcification rates were reduced most under increased temperature and elevated pCO2 conditions (Reynaud et al. 2003). Similarly, juvenile stages of corals were most affected by a combination of temperature and pCO2 increase (Anlauf et al. 2011). In adult corals, increased pCO2 can reduce vulnerability to temperature stress such as bleaching (Anthony et al. 2008). In echinoid larvae, increased temperature and acidification act as additive stressors on growth in some species while in others, moderate warming reduces the negative effect of acidification on growth (Sheppard Brennand et al. 2010; Byrne et al. 2011, 2013). For adult asteroids (Parvulastrea exigua), temperature was shown to have a stronger effect compared to hypercapnia, but increased metabolism at higher temperatures was reduced under simultaneous pH stress (McElroy et al. 2012). Similarly, temperature-affected metabolism of ophiuroids and a reduction in metabolism at intermediate pCO2 were observed at two separate temperatures (Christensen et al. 2011). The temperate ophiuroid Ophiura ophiura and the echinoid Paracentrotus lividus both showed an effect of pH on metabolism under low water temperature, but not under high temperature (Wood et al. 2010; Catarino et al. 2012). Combined temperature and pH stress showed no interaction in several response parameters in the polar ophiuroid Ophiocten sericeum, but arm regeneration in an extreme pH treatment (pH 7.3) was hampered under increased temperatures (Wood et al. 2011). The only study investigating interactive effects of temperature and pCO2 in tropical adult echinoids found that increased pCO2 decreased calcification in Echinometra lacunter more in winter than in summer (Courtney et al. 2013).

We investigated the physiological response of the adult rock-boring Indo-Pacific sea urchin Echinometra sp. A in animals acclimated for approximately 10 weeks to near-future OA conditions (~860–940 μATM) and temperature increase (+2° to 3° above present summer average). Echinometra sp. A is one of the most abundant tropical sea urchin species in shallow reef areas where it can assume densities of 100 individuals m−2 (McClanahan and Muthiga 2007). This species is ecologically important because it can control algal growth, and high densities can prevent recovery of fish and coral populations after disturbance (McClanahan et al. 1996). In addition, feeding-driven bioerosion by Echinometra species removes carbonate (Downing and El-Zahr 1987; Carreiro-Silva and McClanahan 2001) which can reduce net accretion on coral reefs. Resulting burrows are often highly visible in the upper reef margins, and the species is thus habitat forming (McClanahan and Muthiga 2007). Growth of small adult E. mathaei was significantly reduced with only a small increase of pCO2 (220 μatm, pH 7.9; Shirayama and Thornton 2005). Similarly, growth of larger adult Echinometra sp. A was slightly reduced over a 7-week period under increased pCO2 (Uthicke et al. 2013). However, echinoids (including Echinometra spp.) at a tropical CO2 vent exhibited no decrease in abundance with decreased pH/increased pCO2 (Fabricius et al. 2014). As characteristic of echinoid echinoplutei (Byrne et al. 2013), increased pCO2 reduces larval growth and increases abnormal development in larval and adult Echinometra spp. (Kurihara and Shirayama 2004; Uthicke et al. 2013).

Only limited work has been conducted on a temperature effect on Echinometra spp. Its wide tropical Indo-Pacific distribution suggests a large temperature tolerance, and it can be found in locations with water temperature variations between 15 and 31 °C (summarised in Muthiga and Jaccarini 2005). However, given its shallow-water habitat, it can be assumed that E. mathaei can withstand extreme temperatures for a short period. Adult mortality of this species has been observed at ~40 °C (Tsuchiya et al. 1987). Echinometra mathaei can still achieve high fertilisation rates at 36 °C, but embryos only develop normally below 34 °C (Rupp 1973). Healthy development to early larval stages occurs between 19 and 31 °C (Rahman et al. 2007).

The main aim of this study was to test the hypothesis that a combined physiological effect of increased pCO2 and temperature is larger than that of each stressor individually. As response parameters, we measured overall growth, respiration, and ammonium excretion of Echinometra sp. A after >70 d of exposure to the respective treatment conditions. In addition, we compared gonad development amongst the treatments and tested for differences in pH and ionic composition of the perivisceral coelomic fluid.

Materials and methods

Specimen collection and experimental set-up

Adult Echinometra sp. A (32–54 mm diameter, 16–68 g wet weight) were collected in September 2011 at 2–5 m water depth at Rib Reef, a midshelf reef in the central section of the Great Barrier Reef (146° 52.49′E, 18° 28.86′S).

Taxonomic confusion exists about the genus Echinometra in the West Pacific Ocean with four species being described (Arakaki et al. 1998). Mitochondrial DNA sequencing of eight individuals from this experiment revealed that the population on Rib Reef belongs to Echinometra sp. A (Landry et al. 2003; GenBank Accession number: KF555645-KF555652). This also applies to individuals from a previous experiment (Uthicke et al. 2013). The size range used represents ‘intermediate-sized’ adult animals at Rib Reef, omitting the smallest and largest specimens.

Sea urchins were placed into the aquaria on the 21 September 2011 (=Day 0) and kept under similar conditions with pCO2 manipulated as described in Uthicke et al. (2013). In short, urchins were fed ad libitum on brown macroalgae (Sargassum spp., Dictyota sp.) and coral rubble encrusted with crustose coralline algae was offered as an additional food source. The experiment was set up in a temperature-controlled (25 ± 1 °C) aquarium room. The aquarium water was filtered through three bag filters (WATERCO, Australia) in series (25, 10, 5 µm).

An AquaMedic (Germany) CO2 dosing system was used to manipulate pCO2 levels. One pH probe (Aqua Medic, accuracy 0.01 pH units) connected to the control system was placed into each of the four header tanks. The two treatments with increased pCO2 concentrations were set to pHNBS 7.90 ± 0.05. This value was chosen to represent pCO2 concentrations likely around the end of this century (RCP 6: 670 ppm, RCP 8.5: 963 ppm; Meinshausen et al. 2011) unless drastic measures such as carbon capture leading to net-carbon reductions are implemented (Arora et al. 2011). Solenoid valves were connected to a standard CO2 cylinder (GE 082, BOC, Australia) with a flow regulator. When the valves opened, CO2 gas was introduced into the header tanks through gas diffuser (AquaMedic).

Temperature in the header tanks was controlled using 4-kW titanium heating bars controlled by a CR 1,000 (Campell Scientific, Australia) control data logger. In the experiment, temperatures were set to 28 °C, the near-average recent summer maximum SST averaged over the warmest months, calculated as 28.71, SD = 0.40, which was determined from the Davies Reef monitoring site (as above) for the ‘present-day’ scenario and 31 °C for the near-future scenario (+2 to +3 °C).

To document potential variation between aquaria temperature and pH (using a temperature-corrected pH meter, OAKTON, USA; pH probe, EUTECH, USA), these were measured manually on most days (N = 55) throughout the experiment.

To confirm target levels and for calculation of pCO2 and saturation states, samples for dissolved inorganic carbon (DIC) and total alkalinity (TA) analyses were taken 4–6 times from each individual aquarium throughout the experiment. These samples were fixed with 125 μL of saturated (7 g in 100 mL) mercuric chloride and analysed using a VINDTA 3C titrator (Marianda, Germany). Alkalinity was analysed by acid titration (Dickson et al. 2007) and DIC by acidification and coulometric CO2 detection (UIC 5105 Coulometer). Calibration was conducted using certified reference seawater (A. G. Dickson, Scripps Institute of Oceanography, Dixon, Batch 106). To describe natural variation of carbon chemistry parameters, samples from two locations with Echinometra sp. A populations were collected several times during different tide levels. The first of these populations was the source population at the backreef of Rib Reef. The second population was on a shallow reef flat on One Tree Island (152° 05.66′E, 23° 30.47′S) in the southern GBR. These samples were fixed and analysed for TA and DIC as described above.

Water from each of the four header tanks was pumped into three 16-L glass treatment aquaria (randomly allocated along the bench space). The flow to each aquarium was regulated via a flow controller set to 450–500 mL min−1. Water flow within each treatment aquarium was enhanced with a small submersible aquarium pump. Each replicate aquarium contained six specimens of Echinometra sp. A.

Light was supplied through 50:50 actinic 420-nm, 10-K trichromatic daylight (Catalina Compact, 12-h dark : 12-h light cycle, similar to field conditions) at 180–200 μmol photons m−2 s−1.

Physiological parameters

Initial wet weight after blotting (±0.1 g) of the urchins was taken on day 1; final weights were taken on the 2nd of December 2011. Thus, growth was measured over 70 d and expressed as the percentage increase of the original weight. To reduce the influence of gut contents on weight, measurements were taken after a 24-h starvation period. The six individuals in each aquarium were distinguishable based on colour patterns and size, which allowed us to calculate individual growth rates.

Respiration rates of individual urchins were measured on day 69 in 15 specimens from each treatment. Respiration was measured in a respirometer that used an OXY-4 (Presens, Germany) fibre-optic oxygen meter to measure oxygen concentration in Perspex chambers (Uthicke et al. 2013). Temperature was set to the treatment temperature and controlled in a water bath (volume: 6.8 L) connected to the pump of a temperature control (±0.1 °C) unit (Lauda, Germany). Optodes inside the chambers were calibrated at the beginning and the end of the measurements using water-saturated air (100 % O2) and water treated with Na2SO4 (0 % O2). Individual Echinometra sp. A were placed into a jar (640 mL) containing the respective treatment water and re-filtered using a 0.5-µm cartridge filter. Specimens were incubated for a minimum of 25 min to allow for an initial period to stabilise temperatures and for individuals to settle. Oxygen saturation never went below 70 %, and a straight slope of the regression line indicated that these levels did not exert stress on the organisms. Blank chambers were run every two runs, but no background respiration was detectable. Respiration rates were calculated from slopes of oxygen concentration over time during the last 15 min of the incubations.

Ammonium excretion of 12 specimens of Echinometra sp. A for each treatment was determined at day 75 during incubations in 500-mL glass jars. Incubation jars were immersed in a flow-through water bath to keep the temperature constant at the treatment temperatures. Similar to the respiration experiments, we used re-filtered (0.5 µm) treatment water for the excretion incubations. Echinoderms can release high amounts of ammonium in the first few minutes of excretion experiments (Uthicke 2001). Therefore, duplicate initial water samples were collected from each incubation jar after a settle period of ~10 min. Subsequently, the jars were closed and duplicate final samples taken at approximately 60 min (53–64 min). Ammonium determination was conducted on an autoanalyser and followed methods as outlined in Ryle et al. (1981). Each specimen was weighed and ammonium excretion rates calculated taking into account actual water volume during incubations (initial volume minus the initial samples and urchin volume). Urchin volume was derived from wet weights using conversions as described in Uthicke et al. (2013).

Oxygen:nitrogen (O:N) ratios were calculated based on the ammonium excretion rates and average oxygen consumption for each treatment combination.

Gonad index and condition, and coelomic fluid composition

At the end of the experiment (07 December 2011, day 77), all Echinometra sp. A were dissected and the coelomic fluid carefully drained. One gonad was placed in Bouin’s fluid for histology, and the other four with remaining tests and organs were oven-dried for 48 h at 60 °C. Dried weight was used in preference to the wet gonad index more commonly used for sea urchins because there can be large inter-individual differences in fluid retention in the gonads. The fixed gonads were processed for routine wax histology, and the gonad sections (7 µm thick) were stained in haematoxylin and eosin. Gonad histological condition was determined by microscopic examination of the sections. The gametogenic state of the gonads was scored in three stages: mature/partly spawned, partly spawned, and post-spawned/spent as in previous studies of sea urchin gonad histology (Byrne et al. 1998). The mature/partly spawned specimens had in the gonad lumen numerous advanced gametes in good condition, whereas the partly spawned gonads had large spaces in the lumen indicative of major gamete release and the post-spawned/spent gonads had degenerating gametes, no gametes, and/or brown lipofuscin pigment deposits.

The pH in the coelomic fluid was measured within 2 min using a pH temperature-corrected pH meter (OAKTON, USA; pH probe: EUTECH, USA). Subsequently, coelomic fluid was filtered (0.45-µM Minisart filter) and stored cold (4 °C) in acid-washed polypropylene vials until measurements (12 d after sampling) of ion concentrations. The volume of the coelomic fluid of a total of 17 individuals was too small for pH and ion concentration measurements, leading to an unbalanced sampling design. Ca, Mg, Na, and K were measured using inductively coupled plasma-atomic emission spectrometry (ICP-AES). For comparison, duplicate water samples were taken from each aquarium and measured following the same procedures as for the urchins. All samples were diluted 1/10 using 0.1 % HNO3 as diluent. IAPSO sea water and NASS-5 were analysed repeatedly throughout the sample runs to ensure reproducibility. Variability over the 3 d of measurements was <2 % for all ions measured.

Statistical analyses

Growth rates, respiration, and ammonium excretion rates (both specimen specific and biomass specific) were dependent on the size of the individuals (regression analysis, p < 0.001). Thus, these data were analysed using analyses of covariance (ANCOVA). The shape of the relationship resembled a power function, and in most cases, using log-transformed-dependent variables provided the best fit (highest R2). Only for biomass-specific excretion rates, the fit was slightly better with the untransformed covariable, and thus, this variable was not transformed in that model. Temperature and pH were regarded as fixed factors, with average (N = 33) readings of temperature and pH from each individual aquarium. Initial tests including a covariate for gonad indices (based on dry weight of the gonads and remainder) showed that this parameter was independent of the urchin weight for the weight range of the experimental animals. Thus, gonad index was analysed with two-factor analyses of variance (ANOVAs). To account for the appropriate error structure, we tested whether ‘aquarium’ included in the model as a random nested effect was significant. We retained this factor in all cases where initial test showed p values for ‘aquarium’ of < 0.25. Nested ANCOVAS and ANOVAS were run as linear mixed-effect models in R. Histological data of gonad condition were scored into three stages and thus consist of ordinal data. These data were analysed with cumulative link models using the ‘ordinal’ library in R.

Interaction terms with p > 0.25 were removed from the models to increase power of the main effects. Assumptions of normality and homogeneity of variances were checked using box plots and residual plots. No deviations from these assumptions were detected in transformed (see results) data. In ANCOVAs, the assumption for homogeneity of slopes was tested by including an interaction between the covariate and the two factors into initial model runs. If this interaction was not significant (the slopes of individual groups were not significantly different), the interaction term was removed. Type III sums of squares were chosen for ANOVAs with unbalanced designs. All statistical analyses were performed in R (R Development Core Team 2012).

Results

We measured DIC and TA on several occasions at the source population and near a shallower reef flat population in the southern GBR (Electronic Supplementary Material, ESM, Table 1). Depending on tide level and time of the day, pCO2 at the source population varied between 316 and 515 µAtm. However, due to the remote location and inaccessibility of the site at night-time, measurements during low tides (expected to yield the highest pCO2) could not be obtained. Data from One Tree Island illustrate that pCO2 may exceed 1,000 µAtm during those situations and can be as low as 240 µAtm during periods of high productivity during the day (ESM Table 1). The sea surface temperature at the time of collection in September was 24–25 °C and was 23–24 °C for the previous month. In situ temperature increased in comparison with our control temperature during the course of the experiment. Long-term water temperatures for this region are available (at: http://data.aims.gov.au/aimsrtds/latestreadings.xhtml, see also Collier et al. 2012; Lamare et al. 2014) from a 4-m deep station at Davies Reef (18° 49.8′S, 147° 37.8°E), a similar midshelf reef to Rib Reef.

Table 1 Analysis of covariance for the growth of Echinometra sp. A in two temperature and two pCO2 treatments
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Temperatures and pH values in each individual treatment tank were very close to target values (averages for aquaria and Tanks given in ESM Table 2). pCO2 values of the ‘near-future’ treatment were in the range expected under RCP 6 to RCP 8.5.

Growth and metabolism

Most of the Echinometra sp. A (66/72) exhibited measurable growth during the 70-d measuring period, and the weight of the remainder did not change throughout the experiment. Temperature and pH alone had no significant effects on growth rates (ANCOVA; Table 1; Fig. 1a). However, the interaction between temperature and pH was marginally significant (Table 1), and a plot of each individual treatment group illustrated that the largest reduction in growth occurred under elevated pCO2 and elevated temperatures (49.5 % reduction compared to 28 °C/pH 8.1; Fig. 1b).

Fig. 1
figure 1

Growth of Echinometra sp. A (as percentage of initial weight) affected by a temperature and b the interaction between temperature and elevated pCO2. Boxes for 28 °C are filled grey, 31° white. Whiskers denote 1.5 × the inter-quartile range, the black line indicates the mean

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Respiration was measured in 60 specimens on day 69. No effects of the main factors were detected in standard metabolic rate (SMR = respiration per unit body mass; Table 2), but the interaction between temperature and pCO2 was marginally significant. This was caused by the highest respiration rates being measured in the high temperature/high pCO2 treatment (Fig. 2). However, the value in the latter treatment was only 5.9 % above that for the present day ‘control’ treatment.

Table 2 Analysis of covariance for respiration of Echinometra sp. A in two temperature and two pCO2 treatments
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Fig. 2
figure 2

Respiration of Echinometra sp. A as standard metabolic rate a standardised per gram of wet weight of the specimens or b per specimen, and differences caused by different-sized urchins were modelled out for statistical analysis (see Table 2). Boxes for 28 °C treatments are filled grey, 31 °C white. Whiskers denote 1.5 × the inter-quartile range, and the black line indicates the mean

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Respiration per specimen was significantly higher in the high temperature treatment (Table 2; Fig. 2b). However, for this parameter, the interaction term was also marginally significant. The combined stressor (high temperature/high pCO2) treatment was 22.1 % above the value for the control.

Ammonia excretion rates from 12 specimens per treatment were measured on day 75 of the experiment. Weight-specific ammonium excretion was elevated in the 31 °C treatment (average 44.80 nmol NH4 h−1, SE = 2.27 nmol NH4 h−1; Table 3) compared to the 28 °C (average 39.43 nmol NH4 h−1 g−1, SE = 2.06 nmol NH4 h−1), but this effect was only marginally significant. Per specimen values were significantly higher in the elevated temperature treatment (31 °C average 1.70 µmol NH4 h−1, SE = 0.08 µmol NH4 h−1; 28 °C average 1.40 µmol NH4 h−1, SE = 0.08 µmol NH4 h−1; Table 3). Similar to respiration rates, both excretion parameters showed highest rates at the high temperature/high pCO2 treatment (Fig. 3). However, the interaction term of the ANCOVA was not significant for weight-specific rates and specimen-specific rates (Table 3).

Table 3 Analysis of covariance for excretion of Echinometra sp. A in two temperature and two pCO2 treatments
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Fig. 3
figure 3

Ammonium excretion rates of Echinometra sp. A as a weight-specific rate (standardised per gram of wet weight of the specimens) or b per specimen, and differences caused by different sized urchins were modelled out for statistical analysis (see Table 3). Boxes for 28 °C treatments are filled grey, 31 °C white. Whiskers denote 1.5 × the inter-quartile range, and the black line indicates the mean

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There was no significant difference between O:N ratios caused by the temperature or pH treatment (Table 3), with an overall mean value of 44.4 (1 SE = 1.56, N = 48).

Gonad index and histological condition

Gonad indices were not significantly different between pH treatments, and temperature by pH interactions was not significant for all individuals pooled, males or females (Table 4). In contrast, gonad indices were higher in the lower temperature treatment, and this was significant for all individuals pooled and for males (Fig. 4; Table 4).

Table 4 Analysis of variance for gonad indices of Echinometra sp. A in two temperature and two pCO2 treatments
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Fig. 4
figure 4

Gonad index (based on dry body weight and dry gonad weight of Echinometra sp. A (a all individuals, b males, c females). Boxes for 28 °C treatments are filled grey, and 31 °C treatments in white. Whiskers denote 1.5 × the inter-quartile range, and the black line indicates the mean

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Gonad histological condition differed amongst treatments (Figs. 5, 6). For the females, there was a marginally significant effect of temperature and the interaction of temperature and pH on the distribution of histological stages (Table 5). Ovaries from urchins maintained in control conditions had abundant mature eggs (Fig. 5) and were in the mature or partly spawned condition. With increased temperature and decreased pH, ovary condition declined with an increase in the number of individuals in the post-spawned or spent condition (Fig. 6). These ovaries had few or no eggs in the lumen, and many had degenerating eggs and accumulations of lipofuscin pigment granules (Fig. 5). No females in the control treatment were in the post-spawned category.

Fig. 5
figure 5

Histology of the gonads of female (a–d) and male (e–h) Echinometra sp. A held in experimental treatments for 77 d. The gonads of urchins maintained in control conditions had an abundance of mature gametes (a, e) or were partly spawned (b, f). In the other treatments, the gonads were post-spawned/spent (c, d, g, h) with empty gonad tubules and lipofuscin-like pigment accumulations (e.g., in centre of c). Scale bars = 100 µm

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Fig. 6
figure 6

Histological condition of the female ovaries (top) and male testes (bottom) of Echinometra sp. A maintained in experimental treatments for 77 d

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Table 5 Cumulative link model analysis for ordinal data for female (total N = 34) and male (N = 35) Echinometra sp. A
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Differences in histological categories were more distinct in the males, with the effect of pH/high pCO2 and the interaction being significant (Table 5). With the exception of the high temperature and low pH/high pCO2 treatment, ~50 % of the testes were mature. Testis condition clearly declined in the low pH treatments (Figs. 5, 6), and most distinctly so in the high temperature low pH treatment, where no mature gonads were detected. The latter finding agrees with the significant interaction between the two stressors seen with statistical analysis, suggesting a synergistic effect.

Coelomic fluid

The pH of the coelomic fluid was lower than in the external treatment water of the aquaria. Although the coelomic fluid from Echinometra sp. A from the control pH 8.1 treatment was higher (mean pH = 7.56, SD = 0.05) than that from the urchins kept at pH 7.9 (mean pH = 7.52, SD = 0.07), this effect was not statistically significant (Table 6). Temperature had also no measurable effect on coelomic fluid pH (Table 6).

Table 6 Analysis of variance pH and ion concentrations in the coelomic fluid of Echinometra sp. A in two temperature and two pCO2 treatments
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There was less variation in the ion concentration of samples from the aquarium water (Fig. 7) compared to the concentration in the coelomic fluid samples. No significant difference between water samples of the treatments was detected for any of the four ions measured (2-factor ANOVA, p > 0.1 for each factor and the interaction term). Compared to the external medium, calcium, magnesium and sodium were significantly decreased (indicated by non-overlapping standard deviations, Fig. 7). Potassium was in the same range in the coelomic fluid and in the aquarium water.

Fig. 7
figure 7

Ion concentration in the coelomic fluid of Echinometra sp. A. Treatment levels (Temperature/pH) are listed on the horizontal axes. Boxes for 28 °C treatments are filled grey and, 31 °C white. Horizontal lines represent the mean (solid line) and standard deviation (dashed line) of the average aquarium water, and exact values for these were 413 mg L−1 (1 SD = 5 mg L−1) for Ca, 389 mg L−1 (1 SD = 5 mg L−1) for K, 1,323 mg L−1 (1 SD = 11 mg L−1) for Mg, 1.07 mg L−1 (1 SD = 0.01 mg L−1) for Na

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Internal concentrations of calcium, magnesium and sodium exhibited no differences between temperature or pH treatments (Table 6; Fig. 7). ANOVA for potassium had a marginally significant interaction term between temperature and pH, with values at the 28/7.9 and 31/8.1 treatments elevated above the other treatments (Fig. 7). However, values within these groups are also more variable, and averages only vary by a small amount (7–8 %) between treatments.

Discussion

Our experiments showed that some of the basic metabolic parameters (growth, respiration, ammonium excretion) of the ecologically important sea urchin Echinometra sp. A responded to temperature and/or pH changes. Growth at the high temperature/high pCO2 treatment was reduced, whereas respiration and ammonium excretion increased in that treatment compared to the ‘present-day’ control. Our initial hypothesis that the interaction of increased temperature and pCO2 at levels expected by the end of this century under most representative concentration pathways (Meinshausen et al. 2011) leads to higher stress than the individual stress alone can thus be accepted. However, our study also illustrated that the effects on adults are not large, at least over the 10-week time scale of our study, and in several cases only marginally significant. Echinometra sp A. and other shallow-water reef species are acclimatised to the large (nearly from 250 to 1,300 µmol) daily changes in pCO2 that they experience over the tidal cycle. However, during less extreme tides and in deeper water, fluctuations in pCO2 were much smaller.

Metabolism

We previously showed that the growth of Echinometra sp. A (addressed as E. mathaei) subtly decreased in response to decreased pH/increased pCO2 (Uthicke et al. 2013), which was in agreement with other data on Echinometra species in Japan (Shirayama and Thornton 2005). Although the response to increased pCO2/decreased pH is not always linear, all four other echinoid species thus far investigated can be expected to exhibit reduced growth in near-future OA conditions (Ries et al. 2009; Albright et al. 2012; Stumpp et al. 2012b). In the present experiment, the relatively mild change in pCO2/pH alone did not result in significantly reduced growth. A pCO2 change of similar magnitude applied in Shirayama and Thornton (2005) only resulted in differences in size after longer (>12 weeks) experimental periods. In our study, the largest reduction was seen when temperature was elevated and pH decreased/pCO2 increased.

As for most poikilothermic invertebrates, echinoid metabolism follows the Q10 rule and increases with temperature increase (Ulbricht and Pritchard 1972). This was also observed here for respiration and ammonium excretion, although the increase in respiration was subtle and not significant. For both parameters, the highest metabolic rate was observed in the high temperature/high pCO2 treatment.

Responses of echinoderm respiration to pH changes vary widely and often deviate from the assumption that low pH/hypercapnia induces metabolic depression in marine invertebrates (Pörtner et al. 2004; Pörtner 2008). In a previous study, we showed that respiration in Echinometra sp. A can be subtly reduced under intermediate pCO2 conditions (785–1,261), but were at the same level as the control at 1,786 μATM CO2 (Uthicke et al. 2013). A similar pattern was also observed for temperate ophiuroids and asteroids (Christensen et al. 2011; McElroy et al. 2012). Two boreal-temperate ophiuroid species exhibited increased metabolic rates with decreasing pH (Wood et al. 2008, 2011). In the present study, pH had a slightly enhancing effect on respiration, but this was more distinct under elevated temperatures.

Ammonium excretion rates in tropical holothurians in summer were also higher than those at colder temperatures (Uthicke 2001). A previous study on a temperate echinoid species detected clearly higher ammonium excretion rates under elevated pCO2, but only under treatment conditions much higher than used here (~2,800 uATM; Stumpp et al. 2012b). The latter study also observed reduced O:N ratios, which was interpreted as higher protein catabolism as an additional mechanism for removing protons. Reduced O:N ratios are generally interpreted as a shift in substrate for oxidative metabolism, e.g., towards monoamino dicarboxylic amino acids (Langenbuch and Pörtner 2002). O:N ratios in our study were high, as expected for herbivorous echinoids (Otero-Villanueva et al. 2004), but showed no difference between treatments.

Several studies have investigated the interactive effects of temperature and pH on echinoderm metabolism. Low pH only leads to metabolic upregulation in Ophiura ophiura when in combination with a low temperature treatment (Wood et al. 2010). However, due to lower saturation of calcite, the high temperature/low pH treatment of that study required relocation of energy to maintain calcification. The temperate echinoid Paracentrotus lividus also only increased metabolism with reduced pH under simultaneous reduction of temperature (Catarino et al. 2012). In contrast to the latter study, several parameters measured in Ophiocten sericeum showed no interactive effects between pH and temperature; only arm regeneration was reduced when subjected to extreme low pH in combination with temperature stress (Wood et al. 2011). Also Ophionereis shayeri exhibited interactive effects on temperature and pH on respiration (Christensen et al. 2011). In that study, specimens kept at the lowest pH (7.6) showed a smaller reduction in respiration when held in lower temperature than control and intermediate pH specimens (pH 8.2 and 7.8). As for most studies mentioned above, temperature had a stronger effect on respiration of the asteroid Parvulastra exigua than reduced pH/increased pCO2 (McElroy et al. 2012). However, under the lowest pH treatment, temperature increase above 21 °C does not further increase respiration, in the latter study.

Gonads

Increased temperature (+3 °C) resulted in a decrease in the gonad index, which was most pronounced (and significant) for the males. No effect of pH could be detected although in a previous study, we observed that males held under similar pH conditions as used here had a reduced ability to spawn (Uthicke et al. 2013). An impact on female fecundity after 4 months of exposure to elevated pCO2 was also observed in Strongylocentrotus droebachiensis, but this effect was not present in animals acclimated for longer periods (Dupont et al. 2012). After an exposure of 9 months, individuals of Hemicentrotus pulcherrimus exposed to elevated pCO2 delayed spawning and gonad development by 1 month (Kurihara et al. 2013). Histological examination indicated that the gonads of most of the Echinometra sp. A held under conditions other than the control had fewer mature gametes. For the females, gonad condition indicated degeneration/resorption of the eggs or differential gamete release in high temperature and low pH treatments. For the males, decline in gonad condition was seen in the low pH treatments, and a synergistic effect of the stressors pH and temperature was suggested. It seems that both females and males may have released gametes over the incubation period. Spawning, however, was not observed and may have occurred at night. The urchins from the control tanks are likely to have continued to recruit gametes to the lumen (supported by the feeding regime) in continual gamete development as occurs over the long approximately 5- to 6-month spawning period of this urchin on the GBR (M Byrne pers. obs.). In contrast, gonads of urchins in treatments with elevated temperature and/or elevated pCO2 were unable to generate new cohorts of gametes.

The response of the gonads to high temperature/low pH conditions may have been an acute disturbance response rather than a response on gonad development. However, given the difference observed between the treatments, gonad condition is also an indicator of stress on the metabolism of the urchins. The differences in the response of gonad development in experimental treatments, based on data from weight and histological condition, show that the use of a gonad index can only provide a proxy of organism response, as shown in other studies of sea urchin reproduction (Byrne 1990; Byrne et al. 1998). In order to determine the reproductive response of sea urchins and other organisms to ocean change conditions, it will be important to maintain the brood stock in experimental conditions from the outset of gonad development cycle or conduct studies in organisms under naturally elevated pCO2 such as those on vent systems (Hall-Spencer et al. 2008; Fabricius et al. 2011).

The boreal-temperate echinoid Strongylocentrotus droebachiensis exhibited reduced gonadal growth in treatments with >1,000 µATM pCO2 compared to controls (Stumpp et al. 2012b). Several studies examined gonad development in echinoids under different temperatures from an aquaculture perspective. For instance, gonads of the S. droebachiensis showed little differences in development in two treatments differing by 9 °C temperature (Garrido and Barber 2001). In contrast, gonadal growth in the temperate species Paracentrotus lividus was reduced when temperature exceeded 22 °C (ambient = up to 24 °C in summer; Shpigel et al. 2004). In the latter studies and for the data presented here, it is possible that higher energy demand through faster metabolism at higher temperatures reduced energy availability for gonad development. These responses may also reflect the general trend in ectotherms ascribed to the temperature size rule, with facilitation of growth by moderate warming up to thermal limits where growth decreases (Sheridan and Bickford 2011).

Coelomic fluid

Amongst echinoderms, echinoids have the highest buffer capacity of the coelomic fluid to ambient pCO2 increase (Collard et al. 2013). The intestine of these animals can act as a barrier for bicarbonate ions and is selective for ion diffusion (Holtmann et al. 2013).

Coelomic fluid pH in the tropical species investigated here was at similar low levels as reported for temperate echinoids (Miles et al. 2007; Spicer et al. 2011); more extreme values (down to ~pH 7.2) were observed during emersion (Burnett et al. 2002).

Coelomic fluid pH of S. droebachiensis decreased significantly (acidosis) after 4 d of exposure to moderately and highly elevated (>1,400 μATM) pCO2, but values recovered after a longer exposure, possibly due to carbonate dissolution of the test (Spicer et al. 2011). Similarly, pH in the coelomic fluid dropped sharply in Psammechinus miliaris after short-term (days) exposure and recovered slightly after longer exposure periods (Miles et al. 2007). Decreased external pH also reduced coelomic fluid pH significantly in Paracentrotus lividus after a 19-d exposure (Catarino et al. 2012). All of the above studies were conducted under distinctly higher pCO2 increase than in the present study. This and the fact that our experiment also constitutes a longer term exposure (77 d) may explain why no significant decrease in pH of the coelomic fluid was observed in Echinometra sp. A.

Miles et al. (2007) described increases in magnesium in the coelomic fluid under increased pCO2 and interpreted this finding as indication for Mg calcite dissolution. Also calcium concentrations in the coelomic fluid can increase with increased pCO2 (Spicer et al. 2011). Magnesium and calcium in the coelomic fluid of Paracentrotus lividus also did not change with external pH, but were altered by temperature (Catarino et al. 2012). In our study, both magnesium and calcium showed no significant differences between treatments, but values of both elements were significantly lower than in the surrounding sea water. This is an indicator that calcification still occurs under mild ocean acidification as mimicked in our experiments. The fact that urchins in all treatments still achieved positive growth supports this interpretation.

Sodium and potassium in S. droebachiensis remained unaffected by increased pCO2 (Spicer et al. 2011), but no comparison was given to the external medium. In the present study, sodium was not affected by treatment, but was also lower than in the aquarium water. It is unclear what caused the apparently erratic pattern in potassium values in the coelomic fluid, with values similar to the external medium in two treatments, and more variation and slightly elevated values in the remaining two (28/7.9 and 31/8.1).

In summary, we detected evidence that interactive effects of likely near-future conditions of elevated SST and elevated pCO2/reduced pH will change the metabolism and have significant negative effects on growth of Echinometra sp A. However, effects were subtle, and ultimately most animals were still able to calcify and grow under those conditions. Densities of adult coral reef echinoids including Echinometra spp. can be equally high or above those of control areas at sites with elevated pCO2 (Fabricius et al. 2014), supporting that the adults of these species may be resilient to increased pCO2. In our experiments, it appears that a higher energy requirement through enhanced metabolism also resulted in less energy available for gonad development. These results support our previous conclusion that the effects of OA on somatic fitness of Echinometra sp. A were smaller than those on reproduction and development (Uthicke et al. 2013), and the latter will have important implications for future survival of the species and population size maintenance.