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Cidaroids spines facing ocean acidification
Marine Environmental Research xxx (xxxx) xxx–xxx
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Cidaroids spines facing ocean acidification Aurélie Derya,∗, Phuong Dat Trana, Philippe Compèreb, Philippe Duboisa a b
Laboratoire de Biologie Marine, Université Libre de Bruxelles, avenue F.D. Roosevelt 50, B-1050 Brussels, Belgium Laboratoire de Morphologie Fonctionnelle et Evolutive, Morphologie ultrastructurale, allée du 6 Août 15, 4000 Liège, Belgium
A R T I C LE I N FO
A B S T R A C T
Keywords: Ocean acidification Benthos Sea urchin Spine Minerals Magnesium concentration Epibionts
When facing seawater undersaturated towards calcium carbonates, spines of classical sea urchins (euechinoids) show traces of corrosion although they are covered by an epidermis. Cidaroids (a sister clade of euechinoids) are provided with mature spines devoid of epidermis, which makes them, at first sight, more sensitive to dissolution when facing undersaturated seawater. A recent study showed that spines of a tropical cidaroid are resistant to dissolution due to the high density and the low magnesium concentration of the peculiar external spine layer, the cortex. The biofilm and epibionts covering the spines was also suggested to take part in the spine protection. Here, we investigate the protective role of these factors in different cidaroid species from a broad range of latitude, temperature and depth. The high density of the cortical layer and the cover of biofilm and epibionts were confirmed as key protection against dissolution. The low magnesium concentration of cidaroid spines compared to that of euechinoid ones makes them less soluble in general.
1. Introduction Anthropogenic atmospheric CO2 dissolves in the ocean and modifies the chemistry of seawater. The main effects are a decrease of the pH and calcium carbonate saturation state, the whole phenomenon being called ocean acidification (OA). These changes might jeopardize organisms building a calcium carbonate skeleton in two ways: (1) by increasing the energetic cost of eliminating protons from the calcification site, thereby reducing growth rate and (2) by inducing the dissolution of the skeletal structures in contact with seawater undersaturated with respect to calcium carbonate. Dissolution depends on the presence of protecting organic layers and on the solubility of the considered calcium carbonate polymorph, aragonite and high-magnesium calcites being the most soluble among crystalline forms. The solubility of magnesium calcite varies according to the amount of magnesium ions substituted to calcium in the calcite lattice. The relationship between solubility of magnesium calcites and magnesium concentrations is debated (Morse and Mackenzie, 1990; Morse et al., 2006; Andersson et al., 2008). Recent developments favored a view that low-magnesium calcites (up to 4 mole % MgCO3) have a solubility equal or close to that of pure calcite while high-magnesium calcites (from 6 mole % MgCO3) have a solubility equal or higher to that of aragonite (Fig. S1 in Lebrato et al., 2016). Furthermore, other factors, as crystal size or other structural characteristics, also influence the solubility of calcite resulting sometimes in large effects in solubility between abiotic and biogenic magnesium calcites (Morse et al., 2006). ∗
Sea urchins build an extensive high-magnesium calcite skeleton and have been considered at risk due to OA. However, recent results question this paradigm, at least in adults (Calosi et al., 2013; Dubois, 2014; Hazan et al., 2014; Uthicke et al., 2014;Collard et al. 2015, 2016; Moulin et al., 2015; Dery et al., 2017). Indeed, the main component of the skeleton (the test encasing the urchin body) appeared as protected from both undersaturated seawater and extracellular fluids (see Dery et al., 2017 and references therein). On the contrary, spines showed evidence of dissolution and reduced fracture force in the same condition, despite the fact that they are separated from seawater by an epidermis (Dery et al., 2017 and references therein). Considering these results, one would expect that spines of cidaroids (the sister clade to all other echinoids, the latter being called euechinoids) which have mature primary spines devoid of epidermis and whose skeleton is in direct contact with seawater (Märkel and Röser, 1983) would even more suffer from undersaturation. These primary spines are composed of three concentric layers: a central zone, the medulla, a median layer and the peripheral cortex. The two central layers are composed of monocrystalline stereom, like in all sea urchins, while the cortex is polycrystalline. Their initial development takes place under an epidermis but the latter disappears after the formation of the cortex. This cortex may be then colonized by a biofilm and epibionts (Märkel and Röser, 1983; Dery et al., 2014) among them many are calcified. When submitted to undersaturated seawater for 3 weeks, the cortex of the cidaroid Prionocidaris baculosa spines showed only few traces of corrosion while the central and median stereom layers were completely corroded
Corresponding author. E-mail address: [email protected] (A. Dery).
https://doi.org/10.1016/j.marenvres.2018.03.012 Received 12 January 2018; Received in revised form 23 March 2018; Accepted 27 March 2018 0141-1136/ © 2018 Elsevier Ltd. All rights reserved.
Please cite this article as: Dery, A., Marine Environmental Research (2018), https://doi.org/10.1016/j.marenvres.2018.03.012
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individuals per species. We selected 11 species from different locations covering a temperature range from 0.8 °C to 29.1 °C (Table 1). Two species were collected at different depth (Cidaris cidaris and Stylocidaris affinis). Two species were collected at the same location but at different depths (Cidaris cidaris and Stylocidaris affinis). Saturation state of calcite was calculated using data from the databases GLODAP (Global Ocean Data Analysis Project) and NOAA (National Centre for environmental information). Spines were cleaned in 3% sodium hypochlorite for 60–90 min and rinsed in 3 successive baths of Ultrapur water (Arium® Pro, Sartorius) and finally air-dried. Each spine was cut transversally at the middle of the shaft in two segments of similar length. The apical segment of the shaft was used for the measurements. The apical extremity of the segment was removed in order to obtain segments of identical length. The segments were placed on Multiclip (Struers) in screw capsules (Rotilabo®) and immerged in 2 successive ethanol100% solutions for 30 min followed by a solution of 1,2-epoxypropane 99% for 30 min and a solution of acetone 99.8% for 60 min. The segments were then embedded in the hard mixture of Agar Low Viscosity Resin (R1078 kit, Agar Scientific). Samples were placed under vacuum for 2 sessions of 60 min and the polymerisation was completed in an oven at 60 °C for 48 h. The embedded segments of spines were cut transversally into 5 mm slices with a diamond saw. The resulting sections were polished with sandpapers of decreasing grit size (P80, P200, P400, P800, P1200, P2400 and P4000) using an automatic polisher Rotopol-2 (Struers, Germany). The final polishing was performed using 1 μm diamond suspension (non-aqueous, 1PS-1MIC, ESCIL, France). The sections were laid on a stub and bridged to the latter by aluminium sticker and quick drying silver paint (AGAR, G302). Elemental energy dispersive X-ray (EDX) analysis was performed in an environmental SEM (FEI ESEM-FEG XL30) operating at 20 kV under low vacuum condition (0.3–0.4 Torr). Spectra were acquired on uncoated polished surfaces by a Bruker 129 eV silicon drift detector of 10 mm2 with an S-UTW window for the detection of light elements. They were treated by the QUANTAX Esprit 2.1 (Bruker, USA). One spectrum was acquired in each of the three concentric zones or layers on the spines cross sections (cortex, median layer and medulla) of each of the three sampled spines (Fig. 1). Spectra showed the presence of carbon, oxygen, sodium, magnesium, silicon, sulphur, chlorine and calcium. The silicon atoms derived
by the same treatment (Dery et al., 2014, P. baculosa wrongly determined as Phyllacanthus imperialis). This resistance was attributed to the significantly lower porosity and magnesium concentration of the cortex compared to the central and median layer and possibly to the coverage of biofilm and epibionts avoiding a direct contact of the cortex with sea water (Dery et al., 2014). However, the latter study was conducted on a single shallow tropical species while cidaroids are present in all oceans and depths with some populations or species living below the saturation horizon of their skeletal mineral (Sewell and Hofmann, 2010; Lebrato et al., 2016). Much too few other data is available to determine if these properties are general adaptative or preadaptive features of this clade or particular to P. baculosa. Slightly lower magnesium concentration in the cortex was reported for the Antarctic species Ctenocidaris speciosa and the temperate Stylocidaris affinis but those differences were not statistically tested (Märkel et al., 1971; Catarino et al., 2013). Interestingly, Catarino et al. (2013) reported significantly lower magnesium concentration in the cortex of spines from C. speciosa collected below the saturation horizon for aragonite (used as a proxy of the saturation horizon for high-magnesium calcite). All other studies reporting Mg concentration in cidaroid spines did not differentiate between cortex and central and median layers (reviewed in Smith et al., 2016 and Lebrato et al., 2016). Epibionts growing on cidaroid spines are generally viewed as parasitic, at least when considered collectively (David et al., 2009) but their real impact has never been addressed beyond bioerosion. Therefore, the aim of the present study was to carry out a comparative study of the cortex magnesium concentration and porosity of a large number of cidaroids species from a broad range of latitudes, temperatures and environments to assess if these characteristics are general adaptive features of the clade. Specimens from different depths of two species were also analyzed. Finally we assessed experimentally the possible protection offered by epibionts and biofilm when the spines face corrosive water. 2. Material and methods 2.1. MgCO3 concentration and density of the different layers of mature primary spines of cidaroids Three mature primary spines were collected from each of three
Table 1 Taxonomy, origin and seawater conditions in collecting sites of used specimens. (*) Collection from the “Université Libre de Bruxelles” (**) collection of the “Muséum national d’Histoire naturelle de Paris” (***) collection of the “Université de Bourgogne – Franche-Comté” at Dijon and (****) individuals kindly provided by Maria Byrne. collection number
species
region
Location
Latitude
Longitude
Temperature
Ωcal
depth
Preservation
sampling
(***) (***) 2013022737 (*) 190810-26; CP2773-01(02) (***) MNHN-IE-2013-16014(15,16) (**) (**) (**)
Ctenocidaris spinosa Homalocidaris gigantea Notocidaris mortenseni Stereocidaris granularis
Antarctic Antarctic Antarctic Tropical
Weddell sea Weddell sea Bransfield strait Papoua-New Guinea
−71.1 −71.1 −62.95 −9.41
−11.46 −11.46 −58.24 160.53
0.8 0.8 1.7 6.3
1.99 1.99 1.88 1.55
277 277 325 537–619
alcohol alcohol alcohol alcohol
2003 2003 2013 2007
Cidaris cidaris
Temperate
Gran Canaria, Spain
31.87
−28.06
12.1
3.39
480
dry
1993
Cidaris cidaris Cidaris cidaris
Temperate Temperate
Gran Canaria, Spain Gran Canaria, Spain
31.87 31.87
−28.06 −28.06
10 8
2.71 2.25
750 1060–1380
dry dry
(*) MNHN-IE-2013-16017(-18,19) (**) MNHN-IE-2013-16007(-8,-9) (**) 100193-01(02,03) (*) (****)
Stylocidaris affinis Stylocidaris affinis
Temperate Temperate
Antiparos, Greace Gran Canaria, Spain
37.01 28.01
25.04 −15.9
16.5 15.3
6.44 3.87
60 215
dry dry
1993 1993 (2) 1987 (1) 2007 1993
Stylocidaris affinis
Temperate
Gran Canaria, Spain
33.98
−30.19
9.9
2.66
665
dry
1993
Temperate Temperate
Gran Canaria, Spain Sydney, Australia
30.11 −33.97
−28.27 151.25
15 17
3.98 5.22
340 1.7
alcohol dry
1993 2013
190810-29; CP2804-01 (***)
Stylocidaris lineata Phyllacanthus parvispinus Stylocidaris bracteata
Tropical
−9.15
161.21
23
4.27
150–175
alcohol
2007
(*)
Eucidaris tribuloides
Tropical
18.47
−77.89
29.1
6.47
10
dry
2012
(*)
Prionocidaris baculosa
Tropical
Malaita, Solomon Islands Montego Bay, Jamaica Philippines
10.21
123.85
28.7
6.16
10
dry
2008
2
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nested in depth) followed by Tukey tests for multiple comparisons using the appropriate mean square error. All statistical analyses were computed according to Doncaster and Davey (2007) using the Systat12 software.
2.2. Epibiont experiment Experiments were carried out on primary spines of Prionocidaris baculosa, purchased from the company De Jong Marinelife (The Netherlands). Specimens originate from the coastal region of Cebu in Philippines. They were kept in an aquarium at 32.5 psμ salinity, pHT 8.1 and 25 °C. They were fed ad libitum with artificial sea urchin food (Zeigler™, USA). Different types of spines were used: (1) mature primary spines covered by a biofilm and epibionts (2) young primary spines (having a well-developed cortex) but still covered by the epidermis and (3) mature primary spines cleaned, to reduce the epibiont cover, in 3% sodium hypochlorite for 30 min and rinsed 3 times with Ultrapur water (Sartorius, Arium®Pro). The spines were removed just before the start of the experiment except for cleaned ones removed the day before. The efficiency of epibiont removal and integrity of the cleaned skeleton was checked by SEM JEOL JSM-7200F. For this purpose, we cleaned six spines from three specimens of P. baculosa (two spines from each individual). Three spines were directly air-dried and the three others were cleaned as previously mentioned. They were mounted on aluminium stubs, coated with gold (JEOL JFC-1100E). The microstructure of the spine was not affected by the treatment with sodium hypochlorite (no traces of corrosion were visible). Nevertheless, the epibiont cover has decreased substantially. Preliminary assays indicated that the optimal incubation time was 12 h, avoiding bacterial growth and degradation of the epidermis present around the young primary spines. Spines were incubated in the dark at 25 °C for 12 h in filtered (0.22 μm, Sterivex™, Millipore) natural seawater at different pHT (8, 7.7, 7.4). The pH of the seawater was manipulated by bubbling CO2 into 1L containers under control of a pH controller system Aquastar (iks ComputerSysteme GmbH, Karlsbad, Germany). Before the start of the experiment, pH and electromotive force were measured with a Metrohm pH-meter (826 pH mobile, combined with a glass electrode (reference 6.0228.010; Metrohm, Darmstadt, Germany) calibrated with CertiPUR® buffer solutions pH 4.00 and 7.00 (Merck, Darmstadt, Germany). All pH measurements were converted to total scale according to DelValls and Dickson's method using TRIS/AMP buffers (kindly provided by the laboratory of “Biogéochimie et Modélisation du Système Terre: Océanographie chimique et Géochimie des Eaux” Prof. Lei Chou, Université Libre de Bruxelles). Total alkalinity (AT) was measured by Gran's titration with HCl 0.1 M containing 0.7 M NaCl using an automatic titrator (718 STAT Titrino Metrohm). Salinity was 32.0 ± 0.1 psμ and temperature was 24.9 ± 0.4 °C. Initial conditions in seawater are reported in Table 2. Spines were placed individually in 12 ml sealed Exetainer tubes (Labco Limited) totally filled with seawater prepared as previously described. For each pHT (8,7.7, 7.4), 3 replicates were used. Exetainer tubes filled with seawater but without spines were used as control. At the end of the experiment AT of the seawater was determined with a potentiometric titration method adapted to small volumes (Collard et al., 2013). Briefly, a potentiometric titration was realized on a 0.5 mL sample by adding first 5 μl of 0.1 M HCl and 0.7 M NaCl (Merck, Darmstadt, Germany) and then 1 μL at a time. After each
Fig. 1. Scanning electron microscopy of a cross section of a mature primary spine of the cidaroid sea urchin Prionocidaris baculosa. c, cortex; e, epibionts; m, medulla; ml, median layer.
from polishing residues and chlorine from the resin as well as a part of carbon and oxygen. The elemental quantitative analyses used an automatic background subtraction and the standard-less ZAF correction matrix to calculate the elemental composition in atomic percent. The MgCO3 mole percentage was calculated in atomic percent (Moureaux et al., 2010). The porosity was assessed through skeleton density. This was determined by analysing backscattered electron images (SEM-BSE) of spine cross-sections using ImageJ software (NIH). It is calculated on spine cross sections, as the ratio of the mineral area (bright on SEM-BSE images) in a layer on the total surface area of the layer. The calculation of the Mg-calcite saturation state developed by Lebrato et al. (2016) is sensitive to mole % MgCO3 above 5, which makes it unusable for our data given the low percentage of magnesium present in the skeleton of cidaroid spines. Therefore, taking into account the “minimally prepared” curve of Plummer and Mackenzie (1974), for low-Mg calcite, the saturation horizon of calcite was used as a proxy of the magnesium calcite saturation state. The saturation state of calcite was calculated using the CO2SYS program. Temperature, salinity, alkalinity and concentration of dissolved inorganic carbon (DIC) where obtained on the database GLODAP (Global Ocean Data Analysis Project) and NOAA (National Centre for environmental Information). Simple linear regression analysis was used to determine interspecific relation between skeletal magnesium concentration or density and seawater temperature and saturation state. Magnesium concentrations and skeletal densities of the different layers of primary spines within a species were compared by a model III ANOVA (random factors: individual, spine nested in individual; repeated crossed fixed factor: layer) followed by Tukey tests for multiple comparisons using the appropriate mean square error (Doncaster and Davey, 2007). Magnesium concentrations and skeletal densities of spines from individuals collected at different depths (Cidaris cidaris and Stylocidaris affinis) were compared for each layer by a model III ANOVA (fixed repeated factor: layer, random factor: spine nested in individual Table 2 Initial physicochemical parameters of the seawater at each pH value. Target pH
salinity
Temperature (°C)
AT (μmol/kg)
pHT
DIC (μmol/kg)
pCO2 (μatm)
[HCO3] (μmol/kg)
[CO3] (μmol/kg)
[CO2] (μmol/kg)
Ωca
Ωarag
8 7.7 7.4
32.0 32.1 32.0
24.5 25.0 25.2
2262 2122 2155
7.96 7.69 7.43
2038 2012 2129
514.6 967.3 1895.1
1857 1891 2020
166.2 93.8 55.0
15.0 27.8 54.2
4.09 2.31 1.36
2.67 1.51 0.89
3
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granularis and S. bracteata (pTukey = 0.078 and pTukey = 0.445 respectively) (Table S3). The density of the different layers of the spine is positively linked to temperature and the saturation state of calcium carbonate (pregression≤0.047) except in the cortex where density is not linked to the saturation state. However, these two parameters poorly explain the variation in density (R2 ≤ 0.21) (Table 3). Density did not differ significantly in regards to depth for the medulla of S. affinis (pANOVA = 0.439) and for C. cidaris (pANOVA = 0.262) (Table S4). However, density differed significantly according to the interaction between the layer and the depth for S. affinis and C. cidaris (pANOVA = 0.025 and 0.026 respectively). The density between the different layers at the same depth is significantly different (pTukey≤0.044) except between the median layer and the medulla at 215 m for S. affinis (pTukey = 0.097). The density of the same layer at different depth does not differ significantly except for the median layer between 480 m and 1060 m for C. cidaris (pTukey = 0.027).
addition, the sample was thoroughly agitated and pH was measured using a 3 mm diameter glass microelectrode (reference 6.0224.100; Metrohm, Darmstadt, Germany). The AT-SW was then calculated using Gran's function (Gran, 1952). Difference in alkalinity after 12 h incubations were computed for each tube and normalized to a cm2 of the surface of primary spine of P. baculosa to be comparable. Normalization was performed by dividing the obtained values by the surface of primary spine approximated to the shape of a cone. The quality of the total alkalinity measurements was checked daily using a certified standard solution (CRM#151) provided by the laboratory of Andrew G. Dickson, Scripps, California, USA). Error in the measurements was 8.6% ± 2.1 (m ± sd, n = 14). Differences in alkalinity were compared by a two factors ANOVA model I (fixed factors: seawater pH, type of spine) followed by Tukey tests for multiple comparisons. All statistical analyses were carried out with Systat12 software. 3. Results
3.3. Epibiont experiment 3.1. Magnesium concentration Dissolution of spines with epibionts or epidermis and with experimentally reduced epibionts cover submitted to different pH was monitored through changes in total alkalinity (AT) in the surrounding seawater. The results are expressed as the variation of total alkalinity (ΔAT) per cm2 of spine compared to values at initial time (t0). The pH and type of spine have a significant effect on the alkalinity variation (pANOVA < 10−3) but the interaction between these two factors is not significant (pANOVA = 0.46) (Table S5). Whatever the type of spine, after 12 h of experiment, the alkalinity increase was significantly higher at pHT 7.4 than at pHT 8.0 (pTukey < 10−3). At pHT 7.7, this increase was intermediate and not significantly different, neither from 8.0 nor 7.4 treatments (although at the margin of significance, pTukey = 0.054 and 0.081, respectively). The type of spine influenced the ΔAT (Fig. 5, Table S5). ΔAT of spines with a reduced epibiont cover was significantly higher than the ΔAT of control and spines covered by epibionts (pTukey < 10−3). The ΔAT was not statistically different between the control and the spines covered by epibionts (pTukey = 0.875) (Fig. 5). The ΔAT of spines covered by a living epidermis did not differ significantly nor from the control and the spines covered by epibionts, neither from the spines with a reduced epibiont cover (pTukey≥0.05).
Magnesium concentrations in the different layers of primary spines of cidaroids are reported in Fig. 2 and Table S1. The concentrations of magnesium differed significantly according to the spine layers (cortex, median layer, medulla) (pANOVA ≤ 0.036) except for 1 species: N. mortenseni (pANOVA = 0.169) (Table S2). The molar percentage of MgCO3 was significantly lower in the cortical layer than in the median layer in most species (pTukey≤0.047, Table S3) except N. mortenseni, H. gigantea, C. spinosa and C. cidaris at 1060 m for which there was no significant difference in magnesium concentration between these two layers (pANOVA = 0.169, pTukey = 0.389, pTukey = 0.05, pTukey = 0.055 respectively). The molar percentages of MgCO3 did no differ significantly between the cortex and the medulla (pTukey≥0.074) except in H. gigantea, S. affinis (665 m), S. lineata for which the magnesium concentration was higher in the cortex (pTukey = 0.028, 0.042 and 0.016 respectively) and P. baculosa for which the magnesium concentration is lower in the cortex (Dery et al., 2014). The molar percentage of MgCO3 was significantly higher in the median layer than in the medulla (pTukey≤0.048) except in N. mortenseni, H. gigantea and S. bracteata (pANOVA = 0.160, pTukey = 0.058 and pTukey = 0.109). The molar percentage of MgCO3 in the cortex was lower or equal to 5% in all species, making this layer a low-magnesium calcite. This concentration was higher than 5% in the median layer of the three tropical species and in the medulla of P. baculosa. The molar percentage of MgCO3 is positively linked to temperature and the respective saturation state of calcium carbonate for each layer of the spine (pregression < 0.001, R2 ≥ 0.542)(Fig. 3, Table 3). However, temperature and saturation state are highly correlated in our data set (R2 = 0.86,P < 10−3). The molar percentage of MgCO3 did not differ significantly in regards to depth or to the interaction between the layer and the depth for both C. cidaris and S. affinis (pANOVA ≥ 0.075)(Table S4).
4. Discussion 4.1. Magnesium concentration In most species analyzed in the present study, the Mg concentration in the cortex was significantly lower that in the median layer but was similar to that in the medulla. This points to the well-known ability of echinoderms, and sea urchins in particular, to modulate the Mg concentration between ossicles but also within a single ossicle (Weber, 1969; Magdans and Gies, 2004; Moureaux et al., 2010; Smith et al., 2016). The mechanisms for such modulation have been linked to differences in the organic matrix of mineralization (Hermans et al., 2011) and to different degrees of mineralization control (Aizenberg et al., 1997). Such mechanisms could account for the differences between the medulla and median layer. On the other hand, the cortex is deposited through a different process of mineralization resulting in a polycrystalline aggregate contrary to the regular stereom, which is monocrystalline (Märkel et al., 1971; Märkel and Röser, 1983). The mineralization process of the cortex is currently uninvestigated. So, it is difficult to assess if it is responsible for the rather low magnesium concentration recorded in this layer. The striking difference in the magnesium concentration of the cortex vs. the
3.2. Density The density of the different layers (i.e. the proportion of mineral in cross sections) is presented in Fig. 4. Density differed significantly between the different spine layers of each species (pANOVA ≤ 0.016) (Table S2). Density was significantly higher in the cortex than in the median layer and medulla in all species (pTukey≤ 0.024 and pTukey≤ 0.018 respectively). Density of the median layer was higher than that of the medulla (pTukey≤ 0.007) except in S.
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Fig. 2. Concentration of Mg (mole%MgCO3) in the different layers (cortex, median layer and medulla) of the mature primary spines in different species of cidaroids (mean ± SD, n = 3). Means sharing the same superscript within the same species are not significantly different (α = 0.05). 5
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Fig. 3. Mole%MgCO3 (a,c,e) and mineral density (b,d,f) according to temperature in the cortex, median layer and medulla of the primary spines of cidaroids. Parameters of the regression lines are presented in Table 3.
median layer raises the question of the conditions in fluids within the spine. Are these similar to that of seawater or would the pCO2 within the spine pore space be higher due to the presence of cells? The only transmission electron microscope study of cidaroid spines did not provide information on mature spine without epidermis (Märkel and Röser, 1983). So, that question remains open. The magnesium concentration in the different layers is highly significantly linked to temperature and saturation state, two variables which are highly correlated and that we cannot disentangle in our data set. The relation between magnesium concentration in calcite skeleton and temperature has been reported from a long time (Clarke and Wheeler, 1922; Chave, 1954; Weber, 1969; McClintock et al., 2011; Smith et al., 2016; Lebrato et al., 2016). This effect has been attributed to faster calcification at higher temperature, allowing a higher trapping of the “crystal poisoning” magnesium (Weber, 1969). However, this theory considers that the skeleton magnesium calcite is crystallized from a solution. There is growing evidence that a transient amorphous calcium carbonate (ACC) phase is first deposited (Politi, 2004) which is stabilized by magnesium ions (Raz et al., 2003) and favours magnesium incorporation into calcite which crystallized subsequently (Loste et al., 2003). In this context, it is noteworthy that the angular coefficients of the linear relations between magnesium concentration and temperature
are very similar (0.12 vs 0.14) for the cortex and the medulla (which share similar magnesium concentration) despite the very different processes of mineralization. Whatever their mineralization processes, it is noteworthy that all layers have a magnesium concentration ≤5 mole%MgCO3, except for the median layer of tropical species. So, the whole spine of most species is low-magnesium calcite, which means a rather low solubility, close to that of pure calcite and which, in this range, does not vary much with magnesium concentration (see Fig. S1 in Lebrato et al., 2016). This suggests that the link between lower magnesium concentration in the cortex and higher resistance to acidification of this layer compared to that of the other layers, as reported by Dery et al. (2014) in the tropical P. baculosa, is maybe not a general feature of the clade. This is further supported by the absence of effect of depth (and saturation state) on the magnesium concentration of the different layers in Cidaris cidaris and Stylocidaris affinis. However, if one compares the magnesium concentration in the different layers of cidaroid spines with that in spines of regular euechinoids from the same region (Fig. 6) one may notice that the magnesium concentration in the spines of temperate euechinoids is significantly higher than the magnesium concentration in all layers of temperate cidaroid spines (p < 10−3). The magnesium concentration in spines of tropical euechinoids is also
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Table 3 Parameters of simple linear regressions between magnesium concentration (mole%MgCO3) or density and temperature or respective saturation state of calcium carbonate for each layer (cortex, median layer and medulla) of primary spines of cidaroids (all species taken together). Dependent variable
R2
Magnesium (cortex) 0.626 0.572 Magnesium (median layer) 0.838 0.724 Magnesium (medulla) 0.671 0.542 Density (cortex) 0.031 0.014 Density (median layer) 0.21 0.191 Density (medulla) 0.103 0.055
Effect
Coefficient
Standard error
t
p
Constant Temperature Constant Omega Constant Temperature Constant Omega Constant Temperature Constant Omega Constant Temperature Constant Omega Constant Temperature Constant Omega Constant Temperature Constant Omega
1.746 0.117 1.151 0.594 2.015 0.197 1.1 0.971 1.223 0.137 0.691 0.642 0.916 0.001 0.917 0.004 0.398 0.005 0.376 0.022 0.219 0.003 0.218 0.012
0.134 0.007 0.185 0.041 0.125 0.007 0.213 0.049 0.135 0.008 0.121 0.05 0.008 0.001 0.011 0.003 0.012 0.001 0.016 0.004 0.013 0.001 0.017 0.004
13.059 16.164 6.235 14.438 16.082 27.936 5.157 19.898 9.028 16.78 3.296 12.78 110.578 2.01 84.491 1.334 32.956 5.86 23.498 5.516 17.249 3.852 12.707 2.747
< 10−3 < 10−3 < 10−3 < 10−3 < 10−3 < 10−3 < 10−3 < 10−3 < 10−3 < 10−3 0.001 < 10−3 < 10−3 0.047 < 10−3 0.185 < 10−3 < 10−3 < 10−3 < 10−3 < 10−3 < 10−3 < 10−3 0.007
2016). More generally, this questions risk assessment approaches, which are only based on solubility of the skeleton.
significantly higher than the magnesium concentration in the cortex and medulla of tropical cidaroid spines (p < 10−3). There is no significant difference between the magnesium content of Antarctic euechinoid spines compared to those of cidaroids (p = 0.103) but this is probably due to the low number of data available given a low power of the test. So, the cidaroid clade as a whole has apparently a lower magnesium concentration in spines than euechinoids of the same region.
5. Conclusions The dense cortical layer together with the biofilm and epibionts cover effectively protect “naked” cidaroid spines. The magnesium concentration does not seem to be the most important factor preventing the cortex from dissolution compared to the median layer and medulla. However, the low magnesium concentration of cidaroid spines compared to that of euechinoids suggests that cidaroid spines are less soluble in general. This may account for the occurrence of the clade below the saturation horizon and for its surviving the Permian-Triassic transition during which a severe acidification took place (Twitchett and Oji, 2005). However, it should be kept in mind that the protective effect of the epibionts may vary according to their nature. Perforating epibionts may favour the dissolution of the mineral by increasing the surface exposed to seawater.
4.2. Density In all species investigated, the cortex has a density almost twice that of the two other layers, with less than 20% porosity. This is due to its morphogenesis, proceeding through successive addition of mineral layers while the stereom forms by successive branching of thin trabeculae (Heatfield, 1971, Dery et al., 2014, Gorzelak et al., 2014, 2017). This results in a much lower surface-volume ratio, slowing down the dissolution process. Furthermore, the crystallites making the cortex are not in crystallographic continuity, which would further slow the dissolution process. The high density of the cortex appears as a general feature of the clade and is very probably an important protection against dissolution.
Acknowledgments A. Dery is a Research Assistant of the Université Libre de Bruxelles and Ph. Dubois is a Research Director of the National Fund for Scientific Research (FRS-FNRS; Belgium). The study was supported by FNRS (grant number J.0219.16 SOFTECHI). We benefited from a “credit FNRS Grand Equipement” number U600415F. We would like to thank the “Laboratoire de Biologie des Organismes Marins et Biomimétisme” of Mons for its help in the classical SEM observations and the “Cellule d’Appui à la Recherche et à l’Enseignement en Microscopie, CAREM” of the University of Liege for providing access to environmental SEM with EDX. We thank Dr Lebrato for his advices on saturation state calculation and S.Martin for comments on the epibionts experiment. We thank the “Museum national d’Histoire Naturelle, Paris” and particularly M. Eléaume, the “Université de Bourgogne Franche-Comté, Dijon” and particularly T. Saucède, Chantal De Ridder and Maria Byrne for the help in obtaining samples of sea urchin spines. We would also like to thank all the staff of the Discovery Bay Marine Laboratory, Jamaica, for their welcome and help. This is contribution 790 of the Discovery Bay Marine Lab, U.W.I.
4.3. Epibionts Isolated spines, whose biofilm and a part of their epibionts were removed by bleaching, showed a significantly higher dissolution (as evidenced by alkalinity increase in solution), when exposed to low pH seawater, than mature spines covered by epibionts. Because these spines were bleached, the alkalinity increase cannot be attributed to ammonium production by catabolism. Furthermore, epibionts and epidermis covered spines did not show a significantly higher alkalinity increase dissolution compared to controls. This further supports a low or negligeable ammonium production during the 12 h of incubation. So, the biofilm/epibionts cover and to a lower extent the epidermis seem to have a protective effect against the dissolution of the mineral when exposed to low pH seawater. This highlights the importance of organic layers, whatever their origin, to protect biogenic calcium carbonates from dissolution. Such protection has also been reported in other taxa as molluscs (Tunnicliffe et al., 2009; Thomsen et al., 2010, Peck et al., 7
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Fig. 4. Density of the different layers (cortex, median layer and medulla) of the mature primary spines in different species of cidaroids (mean ± SD, n = 3).
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Fig. 5. Difference of alkalinity in seawater between time 12 h and beginning of the incubation experiment of different types of primary spines of cidaroid (control, mature spines covered by epibionts, young spines with cortex covered by an epidermis and mature spines cleaned in 3% sodium hypochlorite to reduce the epibiont cover) (mean ± SD, n = 3) submitted to seawater at pHT 7.4.
Fig. 6. Magnesium concentration (mole%MgCO3; mean ± SD) in the different layers of primary spines of different species of cidaroids (present study) and in the spines of different species of euechinoids (date from the literature, Smith et al., 2016).
Appendix A. Supplementary data
urchins be more resilient to near-future ocean acidification than expected? Global Change Biol. 21, 605–617. Collard, M., Rastrick, S.P.S., Calosi, P., Demolder, Y., Dille, J., Hall-Spencer, J.M., Milazzo, M., Moulin, L., Widdicombe, S., Dehairs, F., Dubois, P., 2016. The impact of ocean acidification and warming on the skeletal mechanical properties of the sea urchin Paracentrotus lividus from laboratory and field observations. ICES J. Mar. Sci. 73, 727–738. David, B., Stock, R.S., De Carlo, F., Hétérier, V., De Ridder, C., 2009. Microstructures of Antarctic cidaroid spines: diversity of shapes and ectosymbiont attachments. Mar. Biol. 156 (8), 1559–1572. Dery, A., Guibourt, V., Catarino, A.I., Compère, P., Dubois, P., 2014. Properties, morphogenesis, and effect of acidification on spines of the cidaroid sea urchin Phyllacanthus imperialis. Invertebr. Biol. 133, 188–199. Dery, A., Collard, M., Dubois, P., 2017. Ocean acidification reduces spine mechanical strength in euechinoid but not in cidaroid sea urchins. Environ. Sci. Technol. 51 (7), 3640–3648. Doncaster, C.P., Davey, A.J., 2007. Analysis of Variance and Covariance. Cambridge University Press, UK 288. Dubois, P., 2014. The skeleton of postmetamorphic echinoderms in a changing world. Biol. Bull. 226, 3. Gorzelak, P., Stolarski, J., Dery, A., Dubois, P., Escrig, S., Meibom, A., 2014. Ultrascale and microscale growth dynamics of the cidaroid spine of Phyllacanthus imperialis revealed by 26Mg labeling and NanoSIMS isotopic imaging. J. Morphol. 275, 788–796. Gorzelak, P., Dery, A., Dubois, P., Stolarski, J., 2017. Sea urchin growth dynamics at microstructural length scale revealed by Mn-labeling and cathodoluminescence imaging. Front. Zool. 14–42. Gran, G., 1952. Determination of the equivalence point in potentiometric titrations. Part II. Analyst 77, 661–671. Hazan, Y., Wangensteen, O.S., Fine, M., 2014. Tough as a rock-boring urchin: adult Echinometra sp. EE from the Red Sea show high resistance to ocean acidification over
Supplementary data related to this article can be found at http://dx. doi.org/10.1016/j.marenvres.2018.03.012. References Aizenberg, J., Hanson, J., Koetzle, T.F., Weiner, F., Addadi, L., 1997. Control of macromolecule distribution within synthetic and biogenic single calcite crystals. J. Am. Chem. Soc. 119, 881–886. Andersson, A., Mackenzie, F., Bates, N., 2008. Life on the margin: implications of ocean acidification on Mg-calcite, high latitude and cold-water marine calcifiers. Mar. Ecol. Prog. Ser. 373, 265–273. Calosi, P., Rastrick, S.P.S., Graziano, M., Thomas, S.C., Baggini, C., Carter, H.A., HallSpencer, J.M., Milazzo, M., Spicer, J.I., 2013. Distribution of sea urchins living near shallow water CO2 vents is dependent upon species acid–base and ion-regulatory abilities. Mar. Pollut. Bull. 73 (2), 470–484. Catarino, A.I., Guibourt, V., Moureaux, C., De Ridder, C., Compère, P., Dubois, P., 2013. Antarctic urchin Ctenocidaris speciosa spines: lessons from the deep. Cah. Biol. Mar. 54 659–655. Chave, K.E., 1954. Aspects of the biogeochemistry of magnesium 1. Calcareous marine organisms. J. Geol. 62, 3. Clarke, F.W., Wheeler, W.C., 1922. The Inorganic Constituents of Marine Invertebrates. Washington Government Printing Office 62. Collard, M., Laitat, K., Moulin, L., Catarino, A., Grosjean, P., Dubois, P., 2013. Buffer capacity of the coelomic fluid in echinoderms. Comp. Biochem. Physiol. A 166, 199–206. Collard, M., De Ridder, C., David, B., Dehairs, F., Dubois, P., 2015. Could Antarctic sea
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Marine Environmental Research journal homepage: www.elsevier.com/locate/marenvrev
Cidaroids spines facing ocean acidification Aurélie Derya,∗, Phuong Dat Trana, Philippe Compèreb, Philippe Duboisa a b
Laboratoire de Biologie Marine, Université Libre de Bruxelles, avenue F.D. Roosevelt 50, B-1050 Brussels, Belgium Laboratoire de Morphologie Fonctionnelle et Evolutive, Morphologie ultrastructurale, allée du 6 Août 15, 4000 Liège, Belgium
A R T I C LE I N FO
A B S T R A C T
Keywords: Ocean acidification Benthos Sea urchin Spine Minerals Magnesium concentration Epibionts
When facing seawater undersaturated towards calcium carbonates, spines of classical sea urchins (euechinoids) show traces of corrosion although they are covered by an epidermis. Cidaroids (a sister clade of euechinoids) are provided with mature spines devoid of epidermis, which makes them, at first sight, more sensitive to dissolution when facing undersaturated seawater. A recent study showed that spines of a tropical cidaroid are resistant to dissolution due to the high density and the low magnesium concentration of the peculiar external spine layer, the cortex. The biofilm and epibionts covering the spines was also suggested to take part in the spine protection. Here, we investigate the protective role of these factors in different cidaroid species from a broad range of latitude, temperature and depth. The high density of the cortical layer and the cover of biofilm and epibionts were confirmed as key protection against dissolution. The low magnesium concentration of cidaroid spines compared to that of euechinoid ones makes them less soluble in general.
1. Introduction Anthropogenic atmospheric CO2 dissolves in the ocean and modifies the chemistry of seawater. The main effects are a decrease of the pH and calcium carbonate saturation state, the whole phenomenon being called ocean acidification (OA). These changes might jeopardize organisms building a calcium carbonate skeleton in two ways: (1) by increasing the energetic cost of eliminating protons from the calcification site, thereby reducing growth rate and (2) by inducing the dissolution of the skeletal structures in contact with seawater undersaturated with respect to calcium carbonate. Dissolution depends on the presence of protecting organic layers and on the solubility of the considered calcium carbonate polymorph, aragonite and high-magnesium calcites being the most soluble among crystalline forms. The solubility of magnesium calcite varies according to the amount of magnesium ions substituted to calcium in the calcite lattice. The relationship between solubility of magnesium calcites and magnesium concentrations is debated (Morse and Mackenzie, 1990; Morse et al., 2006; Andersson et al., 2008). Recent developments favored a view that low-magnesium calcites (up to 4 mole % MgCO3) have a solubility equal or close to that of pure calcite while high-magnesium calcites (from 6 mole % MgCO3) have a solubility equal or higher to that of aragonite (Fig. S1 in Lebrato et al., 2016). Furthermore, other factors, as crystal size or other structural characteristics, also influence the solubility of calcite resulting sometimes in large effects in solubility between abiotic and biogenic magnesium calcites (Morse et al., 2006). ∗
Sea urchins build an extensive high-magnesium calcite skeleton and have been considered at risk due to OA. However, recent results question this paradigm, at least in adults (Calosi et al., 2013; Dubois, 2014; Hazan et al., 2014; Uthicke et al., 2014;Collard et al. 2015, 2016; Moulin et al., 2015; Dery et al., 2017). Indeed, the main component of the skeleton (the test encasing the urchin body) appeared as protected from both undersaturated seawater and extracellular fluids (see Dery et al., 2017 and references therein). On the contrary, spines showed evidence of dissolution and reduced fracture force in the same condition, despite the fact that they are separated from seawater by an epidermis (Dery et al., 2017 and references therein). Considering these results, one would expect that spines of cidaroids (the sister clade to all other echinoids, the latter being called euechinoids) which have mature primary spines devoid of epidermis and whose skeleton is in direct contact with seawater (Märkel and Röser, 1983) would even more suffer from undersaturation. These primary spines are composed of three concentric layers: a central zone, the medulla, a median layer and the peripheral cortex. The two central layers are composed of monocrystalline stereom, like in all sea urchins, while the cortex is polycrystalline. Their initial development takes place under an epidermis but the latter disappears after the formation of the cortex. This cortex may be then colonized by a biofilm and epibionts (Märkel and Röser, 1983; Dery et al., 2014) among them many are calcified. When submitted to undersaturated seawater for 3 weeks, the cortex of the cidaroid Prionocidaris baculosa spines showed only few traces of corrosion while the central and median stereom layers were completely corroded
Corresponding author. E-mail address: [email protected] (A. Dery).
https://doi.org/10.1016/j.marenvres.2018.03.012 Received 12 January 2018; Received in revised form 23 March 2018; Accepted 27 March 2018 0141-1136/ © 2018 Elsevier Ltd. All rights reserved.
Please cite this article as: Dery, A., Marine Environmental Research (2018), https://doi.org/10.1016/j.marenvres.2018.03.012
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individuals per species. We selected 11 species from different locations covering a temperature range from 0.8 °C to 29.1 °C (Table 1). Two species were collected at different depth (Cidaris cidaris and Stylocidaris affinis). Two species were collected at the same location but at different depths (Cidaris cidaris and Stylocidaris affinis). Saturation state of calcite was calculated using data from the databases GLODAP (Global Ocean Data Analysis Project) and NOAA (National Centre for environmental information). Spines were cleaned in 3% sodium hypochlorite for 60–90 min and rinsed in 3 successive baths of Ultrapur water (Arium® Pro, Sartorius) and finally air-dried. Each spine was cut transversally at the middle of the shaft in two segments of similar length. The apical segment of the shaft was used for the measurements. The apical extremity of the segment was removed in order to obtain segments of identical length. The segments were placed on Multiclip (Struers) in screw capsules (Rotilabo®) and immerged in 2 successive ethanol100% solutions for 30 min followed by a solution of 1,2-epoxypropane 99% for 30 min and a solution of acetone 99.8% for 60 min. The segments were then embedded in the hard mixture of Agar Low Viscosity Resin (R1078 kit, Agar Scientific). Samples were placed under vacuum for 2 sessions of 60 min and the polymerisation was completed in an oven at 60 °C for 48 h. The embedded segments of spines were cut transversally into 5 mm slices with a diamond saw. The resulting sections were polished with sandpapers of decreasing grit size (P80, P200, P400, P800, P1200, P2400 and P4000) using an automatic polisher Rotopol-2 (Struers, Germany). The final polishing was performed using 1 μm diamond suspension (non-aqueous, 1PS-1MIC, ESCIL, France). The sections were laid on a stub and bridged to the latter by aluminium sticker and quick drying silver paint (AGAR, G302). Elemental energy dispersive X-ray (EDX) analysis was performed in an environmental SEM (FEI ESEM-FEG XL30) operating at 20 kV under low vacuum condition (0.3–0.4 Torr). Spectra were acquired on uncoated polished surfaces by a Bruker 129 eV silicon drift detector of 10 mm2 with an S-UTW window for the detection of light elements. They were treated by the QUANTAX Esprit 2.1 (Bruker, USA). One spectrum was acquired in each of the three concentric zones or layers on the spines cross sections (cortex, median layer and medulla) of each of the three sampled spines (Fig. 1). Spectra showed the presence of carbon, oxygen, sodium, magnesium, silicon, sulphur, chlorine and calcium. The silicon atoms derived
by the same treatment (Dery et al., 2014, P. baculosa wrongly determined as Phyllacanthus imperialis). This resistance was attributed to the significantly lower porosity and magnesium concentration of the cortex compared to the central and median layer and possibly to the coverage of biofilm and epibionts avoiding a direct contact of the cortex with sea water (Dery et al., 2014). However, the latter study was conducted on a single shallow tropical species while cidaroids are present in all oceans and depths with some populations or species living below the saturation horizon of their skeletal mineral (Sewell and Hofmann, 2010; Lebrato et al., 2016). Much too few other data is available to determine if these properties are general adaptative or preadaptive features of this clade or particular to P. baculosa. Slightly lower magnesium concentration in the cortex was reported for the Antarctic species Ctenocidaris speciosa and the temperate Stylocidaris affinis but those differences were not statistically tested (Märkel et al., 1971; Catarino et al., 2013). Interestingly, Catarino et al. (2013) reported significantly lower magnesium concentration in the cortex of spines from C. speciosa collected below the saturation horizon for aragonite (used as a proxy of the saturation horizon for high-magnesium calcite). All other studies reporting Mg concentration in cidaroid spines did not differentiate between cortex and central and median layers (reviewed in Smith et al., 2016 and Lebrato et al., 2016). Epibionts growing on cidaroid spines are generally viewed as parasitic, at least when considered collectively (David et al., 2009) but their real impact has never been addressed beyond bioerosion. Therefore, the aim of the present study was to carry out a comparative study of the cortex magnesium concentration and porosity of a large number of cidaroids species from a broad range of latitudes, temperatures and environments to assess if these characteristics are general adaptive features of the clade. Specimens from different depths of two species were also analyzed. Finally we assessed experimentally the possible protection offered by epibionts and biofilm when the spines face corrosive water. 2. Material and methods 2.1. MgCO3 concentration and density of the different layers of mature primary spines of cidaroids Three mature primary spines were collected from each of three
Table 1 Taxonomy, origin and seawater conditions in collecting sites of used specimens. (*) Collection from the “Université Libre de Bruxelles” (**) collection of the “Muséum national d’Histoire naturelle de Paris” (***) collection of the “Université de Bourgogne – Franche-Comté” at Dijon and (****) individuals kindly provided by Maria Byrne. collection number
species
region
Location
Latitude
Longitude
Temperature
Ωcal
depth
Preservation
sampling
(***) (***) 2013022737 (*) 190810-26; CP2773-01(02) (***) MNHN-IE-2013-16014(15,16) (**) (**) (**)
Ctenocidaris spinosa Homalocidaris gigantea Notocidaris mortenseni Stereocidaris granularis
Antarctic Antarctic Antarctic Tropical
Weddell sea Weddell sea Bransfield strait Papoua-New Guinea
−71.1 −71.1 −62.95 −9.41
−11.46 −11.46 −58.24 160.53
0.8 0.8 1.7 6.3
1.99 1.99 1.88 1.55
277 277 325 537–619
alcohol alcohol alcohol alcohol
2003 2003 2013 2007
Cidaris cidaris
Temperate
Gran Canaria, Spain
31.87
−28.06
12.1
3.39
480
dry
1993
Cidaris cidaris Cidaris cidaris
Temperate Temperate
Gran Canaria, Spain Gran Canaria, Spain
31.87 31.87
−28.06 −28.06
10 8
2.71 2.25
750 1060–1380
dry dry
(*) MNHN-IE-2013-16017(-18,19) (**) MNHN-IE-2013-16007(-8,-9) (**) 100193-01(02,03) (*) (****)
Stylocidaris affinis Stylocidaris affinis
Temperate Temperate
Antiparos, Greace Gran Canaria, Spain
37.01 28.01
25.04 −15.9
16.5 15.3
6.44 3.87
60 215
dry dry
1993 1993 (2) 1987 (1) 2007 1993
Stylocidaris affinis
Temperate
Gran Canaria, Spain
33.98
−30.19
9.9
2.66
665
dry
1993
Temperate Temperate
Gran Canaria, Spain Sydney, Australia
30.11 −33.97
−28.27 151.25
15 17
3.98 5.22
340 1.7
alcohol dry
1993 2013
190810-29; CP2804-01 (***)
Stylocidaris lineata Phyllacanthus parvispinus Stylocidaris bracteata
Tropical
−9.15
161.21
23
4.27
150–175
alcohol
2007
(*)
Eucidaris tribuloides
Tropical
18.47
−77.89
29.1
6.47
10
dry
2012
(*)
Prionocidaris baculosa
Tropical
Malaita, Solomon Islands Montego Bay, Jamaica Philippines
10.21
123.85
28.7
6.16
10
dry
2008
2
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nested in depth) followed by Tukey tests for multiple comparisons using the appropriate mean square error. All statistical analyses were computed according to Doncaster and Davey (2007) using the Systat12 software.
2.2. Epibiont experiment Experiments were carried out on primary spines of Prionocidaris baculosa, purchased from the company De Jong Marinelife (The Netherlands). Specimens originate from the coastal region of Cebu in Philippines. They were kept in an aquarium at 32.5 psμ salinity, pHT 8.1 and 25 °C. They were fed ad libitum with artificial sea urchin food (Zeigler™, USA). Different types of spines were used: (1) mature primary spines covered by a biofilm and epibionts (2) young primary spines (having a well-developed cortex) but still covered by the epidermis and (3) mature primary spines cleaned, to reduce the epibiont cover, in 3% sodium hypochlorite for 30 min and rinsed 3 times with Ultrapur water (Sartorius, Arium®Pro). The spines were removed just before the start of the experiment except for cleaned ones removed the day before. The efficiency of epibiont removal and integrity of the cleaned skeleton was checked by SEM JEOL JSM-7200F. For this purpose, we cleaned six spines from three specimens of P. baculosa (two spines from each individual). Three spines were directly air-dried and the three others were cleaned as previously mentioned. They were mounted on aluminium stubs, coated with gold (JEOL JFC-1100E). The microstructure of the spine was not affected by the treatment with sodium hypochlorite (no traces of corrosion were visible). Nevertheless, the epibiont cover has decreased substantially. Preliminary assays indicated that the optimal incubation time was 12 h, avoiding bacterial growth and degradation of the epidermis present around the young primary spines. Spines were incubated in the dark at 25 °C for 12 h in filtered (0.22 μm, Sterivex™, Millipore) natural seawater at different pHT (8, 7.7, 7.4). The pH of the seawater was manipulated by bubbling CO2 into 1L containers under control of a pH controller system Aquastar (iks ComputerSysteme GmbH, Karlsbad, Germany). Before the start of the experiment, pH and electromotive force were measured with a Metrohm pH-meter (826 pH mobile, combined with a glass electrode (reference 6.0228.010; Metrohm, Darmstadt, Germany) calibrated with CertiPUR® buffer solutions pH 4.00 and 7.00 (Merck, Darmstadt, Germany). All pH measurements were converted to total scale according to DelValls and Dickson's method using TRIS/AMP buffers (kindly provided by the laboratory of “Biogéochimie et Modélisation du Système Terre: Océanographie chimique et Géochimie des Eaux” Prof. Lei Chou, Université Libre de Bruxelles). Total alkalinity (AT) was measured by Gran's titration with HCl 0.1 M containing 0.7 M NaCl using an automatic titrator (718 STAT Titrino Metrohm). Salinity was 32.0 ± 0.1 psμ and temperature was 24.9 ± 0.4 °C. Initial conditions in seawater are reported in Table 2. Spines were placed individually in 12 ml sealed Exetainer tubes (Labco Limited) totally filled with seawater prepared as previously described. For each pHT (8,7.7, 7.4), 3 replicates were used. Exetainer tubes filled with seawater but without spines were used as control. At the end of the experiment AT of the seawater was determined with a potentiometric titration method adapted to small volumes (Collard et al., 2013). Briefly, a potentiometric titration was realized on a 0.5 mL sample by adding first 5 μl of 0.1 M HCl and 0.7 M NaCl (Merck, Darmstadt, Germany) and then 1 μL at a time. After each
Fig. 1. Scanning electron microscopy of a cross section of a mature primary spine of the cidaroid sea urchin Prionocidaris baculosa. c, cortex; e, epibionts; m, medulla; ml, median layer.
from polishing residues and chlorine from the resin as well as a part of carbon and oxygen. The elemental quantitative analyses used an automatic background subtraction and the standard-less ZAF correction matrix to calculate the elemental composition in atomic percent. The MgCO3 mole percentage was calculated in atomic percent (Moureaux et al., 2010). The porosity was assessed through skeleton density. This was determined by analysing backscattered electron images (SEM-BSE) of spine cross-sections using ImageJ software (NIH). It is calculated on spine cross sections, as the ratio of the mineral area (bright on SEM-BSE images) in a layer on the total surface area of the layer. The calculation of the Mg-calcite saturation state developed by Lebrato et al. (2016) is sensitive to mole % MgCO3 above 5, which makes it unusable for our data given the low percentage of magnesium present in the skeleton of cidaroid spines. Therefore, taking into account the “minimally prepared” curve of Plummer and Mackenzie (1974), for low-Mg calcite, the saturation horizon of calcite was used as a proxy of the magnesium calcite saturation state. The saturation state of calcite was calculated using the CO2SYS program. Temperature, salinity, alkalinity and concentration of dissolved inorganic carbon (DIC) where obtained on the database GLODAP (Global Ocean Data Analysis Project) and NOAA (National Centre for environmental Information). Simple linear regression analysis was used to determine interspecific relation between skeletal magnesium concentration or density and seawater temperature and saturation state. Magnesium concentrations and skeletal densities of the different layers of primary spines within a species were compared by a model III ANOVA (random factors: individual, spine nested in individual; repeated crossed fixed factor: layer) followed by Tukey tests for multiple comparisons using the appropriate mean square error (Doncaster and Davey, 2007). Magnesium concentrations and skeletal densities of spines from individuals collected at different depths (Cidaris cidaris and Stylocidaris affinis) were compared for each layer by a model III ANOVA (fixed repeated factor: layer, random factor: spine nested in individual Table 2 Initial physicochemical parameters of the seawater at each pH value. Target pH
salinity
Temperature (°C)
AT (μmol/kg)
pHT
DIC (μmol/kg)
pCO2 (μatm)
[HCO3] (μmol/kg)
[CO3] (μmol/kg)
[CO2] (μmol/kg)
Ωca
Ωarag
8 7.7 7.4
32.0 32.1 32.0
24.5 25.0 25.2
2262 2122 2155
7.96 7.69 7.43
2038 2012 2129
514.6 967.3 1895.1
1857 1891 2020
166.2 93.8 55.0
15.0 27.8 54.2
4.09 2.31 1.36
2.67 1.51 0.89
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granularis and S. bracteata (pTukey = 0.078 and pTukey = 0.445 respectively) (Table S3). The density of the different layers of the spine is positively linked to temperature and the saturation state of calcium carbonate (pregression≤0.047) except in the cortex where density is not linked to the saturation state. However, these two parameters poorly explain the variation in density (R2 ≤ 0.21) (Table 3). Density did not differ significantly in regards to depth for the medulla of S. affinis (pANOVA = 0.439) and for C. cidaris (pANOVA = 0.262) (Table S4). However, density differed significantly according to the interaction between the layer and the depth for S. affinis and C. cidaris (pANOVA = 0.025 and 0.026 respectively). The density between the different layers at the same depth is significantly different (pTukey≤0.044) except between the median layer and the medulla at 215 m for S. affinis (pTukey = 0.097). The density of the same layer at different depth does not differ significantly except for the median layer between 480 m and 1060 m for C. cidaris (pTukey = 0.027).
addition, the sample was thoroughly agitated and pH was measured using a 3 mm diameter glass microelectrode (reference 6.0224.100; Metrohm, Darmstadt, Germany). The AT-SW was then calculated using Gran's function (Gran, 1952). Difference in alkalinity after 12 h incubations were computed for each tube and normalized to a cm2 of the surface of primary spine of P. baculosa to be comparable. Normalization was performed by dividing the obtained values by the surface of primary spine approximated to the shape of a cone. The quality of the total alkalinity measurements was checked daily using a certified standard solution (CRM#151) provided by the laboratory of Andrew G. Dickson, Scripps, California, USA). Error in the measurements was 8.6% ± 2.1 (m ± sd, n = 14). Differences in alkalinity were compared by a two factors ANOVA model I (fixed factors: seawater pH, type of spine) followed by Tukey tests for multiple comparisons. All statistical analyses were carried out with Systat12 software. 3. Results
3.3. Epibiont experiment 3.1. Magnesium concentration Dissolution of spines with epibionts or epidermis and with experimentally reduced epibionts cover submitted to different pH was monitored through changes in total alkalinity (AT) in the surrounding seawater. The results are expressed as the variation of total alkalinity (ΔAT) per cm2 of spine compared to values at initial time (t0). The pH and type of spine have a significant effect on the alkalinity variation (pANOVA < 10−3) but the interaction between these two factors is not significant (pANOVA = 0.46) (Table S5). Whatever the type of spine, after 12 h of experiment, the alkalinity increase was significantly higher at pHT 7.4 than at pHT 8.0 (pTukey < 10−3). At pHT 7.7, this increase was intermediate and not significantly different, neither from 8.0 nor 7.4 treatments (although at the margin of significance, pTukey = 0.054 and 0.081, respectively). The type of spine influenced the ΔAT (Fig. 5, Table S5). ΔAT of spines with a reduced epibiont cover was significantly higher than the ΔAT of control and spines covered by epibionts (pTukey < 10−3). The ΔAT was not statistically different between the control and the spines covered by epibionts (pTukey = 0.875) (Fig. 5). The ΔAT of spines covered by a living epidermis did not differ significantly nor from the control and the spines covered by epibionts, neither from the spines with a reduced epibiont cover (pTukey≥0.05).
Magnesium concentrations in the different layers of primary spines of cidaroids are reported in Fig. 2 and Table S1. The concentrations of magnesium differed significantly according to the spine layers (cortex, median layer, medulla) (pANOVA ≤ 0.036) except for 1 species: N. mortenseni (pANOVA = 0.169) (Table S2). The molar percentage of MgCO3 was significantly lower in the cortical layer than in the median layer in most species (pTukey≤0.047, Table S3) except N. mortenseni, H. gigantea, C. spinosa and C. cidaris at 1060 m for which there was no significant difference in magnesium concentration between these two layers (pANOVA = 0.169, pTukey = 0.389, pTukey = 0.05, pTukey = 0.055 respectively). The molar percentages of MgCO3 did no differ significantly between the cortex and the medulla (pTukey≥0.074) except in H. gigantea, S. affinis (665 m), S. lineata for which the magnesium concentration was higher in the cortex (pTukey = 0.028, 0.042 and 0.016 respectively) and P. baculosa for which the magnesium concentration is lower in the cortex (Dery et al., 2014). The molar percentage of MgCO3 was significantly higher in the median layer than in the medulla (pTukey≤0.048) except in N. mortenseni, H. gigantea and S. bracteata (pANOVA = 0.160, pTukey = 0.058 and pTukey = 0.109). The molar percentage of MgCO3 in the cortex was lower or equal to 5% in all species, making this layer a low-magnesium calcite. This concentration was higher than 5% in the median layer of the three tropical species and in the medulla of P. baculosa. The molar percentage of MgCO3 is positively linked to temperature and the respective saturation state of calcium carbonate for each layer of the spine (pregression < 0.001, R2 ≥ 0.542)(Fig. 3, Table 3). However, temperature and saturation state are highly correlated in our data set (R2 = 0.86,P < 10−3). The molar percentage of MgCO3 did not differ significantly in regards to depth or to the interaction between the layer and the depth for both C. cidaris and S. affinis (pANOVA ≥ 0.075)(Table S4).
4. Discussion 4.1. Magnesium concentration In most species analyzed in the present study, the Mg concentration in the cortex was significantly lower that in the median layer but was similar to that in the medulla. This points to the well-known ability of echinoderms, and sea urchins in particular, to modulate the Mg concentration between ossicles but also within a single ossicle (Weber, 1969; Magdans and Gies, 2004; Moureaux et al., 2010; Smith et al., 2016). The mechanisms for such modulation have been linked to differences in the organic matrix of mineralization (Hermans et al., 2011) and to different degrees of mineralization control (Aizenberg et al., 1997). Such mechanisms could account for the differences between the medulla and median layer. On the other hand, the cortex is deposited through a different process of mineralization resulting in a polycrystalline aggregate contrary to the regular stereom, which is monocrystalline (Märkel et al., 1971; Märkel and Röser, 1983). The mineralization process of the cortex is currently uninvestigated. So, it is difficult to assess if it is responsible for the rather low magnesium concentration recorded in this layer. The striking difference in the magnesium concentration of the cortex vs. the
3.2. Density The density of the different layers (i.e. the proportion of mineral in cross sections) is presented in Fig. 4. Density differed significantly between the different spine layers of each species (pANOVA ≤ 0.016) (Table S2). Density was significantly higher in the cortex than in the median layer and medulla in all species (pTukey≤ 0.024 and pTukey≤ 0.018 respectively). Density of the median layer was higher than that of the medulla (pTukey≤ 0.007) except in S.
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Fig. 2. Concentration of Mg (mole%MgCO3) in the different layers (cortex, median layer and medulla) of the mature primary spines in different species of cidaroids (mean ± SD, n = 3). Means sharing the same superscript within the same species are not significantly different (α = 0.05). 5
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Fig. 3. Mole%MgCO3 (a,c,e) and mineral density (b,d,f) according to temperature in the cortex, median layer and medulla of the primary spines of cidaroids. Parameters of the regression lines are presented in Table 3.
median layer raises the question of the conditions in fluids within the spine. Are these similar to that of seawater or would the pCO2 within the spine pore space be higher due to the presence of cells? The only transmission electron microscope study of cidaroid spines did not provide information on mature spine without epidermis (Märkel and Röser, 1983). So, that question remains open. The magnesium concentration in the different layers is highly significantly linked to temperature and saturation state, two variables which are highly correlated and that we cannot disentangle in our data set. The relation between magnesium concentration in calcite skeleton and temperature has been reported from a long time (Clarke and Wheeler, 1922; Chave, 1954; Weber, 1969; McClintock et al., 2011; Smith et al., 2016; Lebrato et al., 2016). This effect has been attributed to faster calcification at higher temperature, allowing a higher trapping of the “crystal poisoning” magnesium (Weber, 1969). However, this theory considers that the skeleton magnesium calcite is crystallized from a solution. There is growing evidence that a transient amorphous calcium carbonate (ACC) phase is first deposited (Politi, 2004) which is stabilized by magnesium ions (Raz et al., 2003) and favours magnesium incorporation into calcite which crystallized subsequently (Loste et al., 2003). In this context, it is noteworthy that the angular coefficients of the linear relations between magnesium concentration and temperature
are very similar (0.12 vs 0.14) for the cortex and the medulla (which share similar magnesium concentration) despite the very different processes of mineralization. Whatever their mineralization processes, it is noteworthy that all layers have a magnesium concentration ≤5 mole%MgCO3, except for the median layer of tropical species. So, the whole spine of most species is low-magnesium calcite, which means a rather low solubility, close to that of pure calcite and which, in this range, does not vary much with magnesium concentration (see Fig. S1 in Lebrato et al., 2016). This suggests that the link between lower magnesium concentration in the cortex and higher resistance to acidification of this layer compared to that of the other layers, as reported by Dery et al. (2014) in the tropical P. baculosa, is maybe not a general feature of the clade. This is further supported by the absence of effect of depth (and saturation state) on the magnesium concentration of the different layers in Cidaris cidaris and Stylocidaris affinis. However, if one compares the magnesium concentration in the different layers of cidaroid spines with that in spines of regular euechinoids from the same region (Fig. 6) one may notice that the magnesium concentration in the spines of temperate euechinoids is significantly higher than the magnesium concentration in all layers of temperate cidaroid spines (p < 10−3). The magnesium concentration in spines of tropical euechinoids is also
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Table 3 Parameters of simple linear regressions between magnesium concentration (mole%MgCO3) or density and temperature or respective saturation state of calcium carbonate for each layer (cortex, median layer and medulla) of primary spines of cidaroids (all species taken together). Dependent variable
R2
Magnesium (cortex) 0.626 0.572 Magnesium (median layer) 0.838 0.724 Magnesium (medulla) 0.671 0.542 Density (cortex) 0.031 0.014 Density (median layer) 0.21 0.191 Density (medulla) 0.103 0.055
Effect
Coefficient
Standard error
t
p
Constant Temperature Constant Omega Constant Temperature Constant Omega Constant Temperature Constant Omega Constant Temperature Constant Omega Constant Temperature Constant Omega Constant Temperature Constant Omega
1.746 0.117 1.151 0.594 2.015 0.197 1.1 0.971 1.223 0.137 0.691 0.642 0.916 0.001 0.917 0.004 0.398 0.005 0.376 0.022 0.219 0.003 0.218 0.012
0.134 0.007 0.185 0.041 0.125 0.007 0.213 0.049 0.135 0.008 0.121 0.05 0.008 0.001 0.011 0.003 0.012 0.001 0.016 0.004 0.013 0.001 0.017 0.004
13.059 16.164 6.235 14.438 16.082 27.936 5.157 19.898 9.028 16.78 3.296 12.78 110.578 2.01 84.491 1.334 32.956 5.86 23.498 5.516 17.249 3.852 12.707 2.747
< 10−3 < 10−3 < 10−3 < 10−3 < 10−3 < 10−3 < 10−3 < 10−3 < 10−3 < 10−3 0.001 < 10−3 < 10−3 0.047 < 10−3 0.185 < 10−3 < 10−3 < 10−3 < 10−3 < 10−3 < 10−3 < 10−3 0.007
2016). More generally, this questions risk assessment approaches, which are only based on solubility of the skeleton.
significantly higher than the magnesium concentration in the cortex and medulla of tropical cidaroid spines (p < 10−3). There is no significant difference between the magnesium content of Antarctic euechinoid spines compared to those of cidaroids (p = 0.103) but this is probably due to the low number of data available given a low power of the test. So, the cidaroid clade as a whole has apparently a lower magnesium concentration in spines than euechinoids of the same region.
5. Conclusions The dense cortical layer together with the biofilm and epibionts cover effectively protect “naked” cidaroid spines. The magnesium concentration does not seem to be the most important factor preventing the cortex from dissolution compared to the median layer and medulla. However, the low magnesium concentration of cidaroid spines compared to that of euechinoids suggests that cidaroid spines are less soluble in general. This may account for the occurrence of the clade below the saturation horizon and for its surviving the Permian-Triassic transition during which a severe acidification took place (Twitchett and Oji, 2005). However, it should be kept in mind that the protective effect of the epibionts may vary according to their nature. Perforating epibionts may favour the dissolution of the mineral by increasing the surface exposed to seawater.
4.2. Density In all species investigated, the cortex has a density almost twice that of the two other layers, with less than 20% porosity. This is due to its morphogenesis, proceeding through successive addition of mineral layers while the stereom forms by successive branching of thin trabeculae (Heatfield, 1971, Dery et al., 2014, Gorzelak et al., 2014, 2017). This results in a much lower surface-volume ratio, slowing down the dissolution process. Furthermore, the crystallites making the cortex are not in crystallographic continuity, which would further slow the dissolution process. The high density of the cortex appears as a general feature of the clade and is very probably an important protection against dissolution.
Acknowledgments A. Dery is a Research Assistant of the Université Libre de Bruxelles and Ph. Dubois is a Research Director of the National Fund for Scientific Research (FRS-FNRS; Belgium). The study was supported by FNRS (grant number J.0219.16 SOFTECHI). We benefited from a “credit FNRS Grand Equipement” number U600415F. We would like to thank the “Laboratoire de Biologie des Organismes Marins et Biomimétisme” of Mons for its help in the classical SEM observations and the “Cellule d’Appui à la Recherche et à l’Enseignement en Microscopie, CAREM” of the University of Liege for providing access to environmental SEM with EDX. We thank Dr Lebrato for his advices on saturation state calculation and S.Martin for comments on the epibionts experiment. We thank the “Museum national d’Histoire Naturelle, Paris” and particularly M. Eléaume, the “Université de Bourgogne Franche-Comté, Dijon” and particularly T. Saucède, Chantal De Ridder and Maria Byrne for the help in obtaining samples of sea urchin spines. We would also like to thank all the staff of the Discovery Bay Marine Laboratory, Jamaica, for their welcome and help. This is contribution 790 of the Discovery Bay Marine Lab, U.W.I.
4.3. Epibionts Isolated spines, whose biofilm and a part of their epibionts were removed by bleaching, showed a significantly higher dissolution (as evidenced by alkalinity increase in solution), when exposed to low pH seawater, than mature spines covered by epibionts. Because these spines were bleached, the alkalinity increase cannot be attributed to ammonium production by catabolism. Furthermore, epibionts and epidermis covered spines did not show a significantly higher alkalinity increase dissolution compared to controls. This further supports a low or negligeable ammonium production during the 12 h of incubation. So, the biofilm/epibionts cover and to a lower extent the epidermis seem to have a protective effect against the dissolution of the mineral when exposed to low pH seawater. This highlights the importance of organic layers, whatever their origin, to protect biogenic calcium carbonates from dissolution. Such protection has also been reported in other taxa as molluscs (Tunnicliffe et al., 2009; Thomsen et al., 2010, Peck et al., 7
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Fig. 4. Density of the different layers (cortex, median layer and medulla) of the mature primary spines in different species of cidaroids (mean ± SD, n = 3).
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Fig. 5. Difference of alkalinity in seawater between time 12 h and beginning of the incubation experiment of different types of primary spines of cidaroid (control, mature spines covered by epibionts, young spines with cortex covered by an epidermis and mature spines cleaned in 3% sodium hypochlorite to reduce the epibiont cover) (mean ± SD, n = 3) submitted to seawater at pHT 7.4.
Fig. 6. Magnesium concentration (mole%MgCO3; mean ± SD) in the different layers of primary spines of different species of cidaroids (present study) and in the spines of different species of euechinoids (date from the literature, Smith et al., 2016).
Appendix A. Supplementary data
urchins be more resilient to near-future ocean acidification than expected? Global Change Biol. 21, 605–617. Collard, M., Rastrick, S.P.S., Calosi, P., Demolder, Y., Dille, J., Hall-Spencer, J.M., Milazzo, M., Moulin, L., Widdicombe, S., Dehairs, F., Dubois, P., 2016. The impact of ocean acidification and warming on the skeletal mechanical properties of the sea urchin Paracentrotus lividus from laboratory and field observations. ICES J. Mar. Sci. 73, 727–738. David, B., Stock, R.S., De Carlo, F., Hétérier, V., De Ridder, C., 2009. Microstructures of Antarctic cidaroid spines: diversity of shapes and ectosymbiont attachments. Mar. Biol. 156 (8), 1559–1572. Dery, A., Guibourt, V., Catarino, A.I., Compère, P., Dubois, P., 2014. Properties, morphogenesis, and effect of acidification on spines of the cidaroid sea urchin Phyllacanthus imperialis. Invertebr. Biol. 133, 188–199. Dery, A., Collard, M., Dubois, P., 2017. Ocean acidification reduces spine mechanical strength in euechinoid but not in cidaroid sea urchins. Environ. Sci. Technol. 51 (7), 3640–3648. Doncaster, C.P., Davey, A.J., 2007. Analysis of Variance and Covariance. Cambridge University Press, UK 288. Dubois, P., 2014. The skeleton of postmetamorphic echinoderms in a changing world. Biol. Bull. 226, 3. Gorzelak, P., Stolarski, J., Dery, A., Dubois, P., Escrig, S., Meibom, A., 2014. Ultrascale and microscale growth dynamics of the cidaroid spine of Phyllacanthus imperialis revealed by 26Mg labeling and NanoSIMS isotopic imaging. J. Morphol. 275, 788–796. Gorzelak, P., Dery, A., Dubois, P., Stolarski, J., 2017. Sea urchin growth dynamics at microstructural length scale revealed by Mn-labeling and cathodoluminescence imaging. Front. Zool. 14–42. Gran, G., 1952. Determination of the equivalence point in potentiometric titrations. Part II. Analyst 77, 661–671. Hazan, Y., Wangensteen, O.S., Fine, M., 2014. Tough as a rock-boring urchin: adult Echinometra sp. EE from the Red Sea show high resistance to ocean acidification over
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