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Article

Ocean Warming Amplifies the Effects of Ocean Acidification on Skeletal Mineralogy and Microstructure in the Asterinid Starfish Aquilonastra yairi

by
Munawar Khalil
1,2,3,*,
Steve S. Doo
1,4,
Marleen Stuhr
1 and
Hildegard Westphal
1,2,4
1
Leibniz Centre for Tropical Marine Research (ZMT), 28359 Bremen, Germany
2
Faculty of Geosciences, University of Bremen, 28359 Bremen, Germany
3
Department of Marine Science, Faculty of Agriculture, Universitas Malikussaleh, Reuleut Main Campus, Aceh 24355, Indonesia
4
Physical Science and Engineering (PSE), Red Sea Research Center (RSRC), King Abdullah University of Science and Technology (KAUST), Thuwal 23955-6900, Saudi Arabia
*
Author to whom correspondence should be addressed.
J. Mar. Sci. Eng. 2022, 10(8), 1065; https://doi.org/10.3390/jmse10081065
Submission received: 31 May 2022 / Revised: 30 June 2022 / Accepted: 1 July 2022 / Published: 3 August 2022
(This article belongs to the Special Issue The Effect of Ocean Acidification on Skeletal Structures)
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Abstract

:
Ocean acidification and ocean warming compromise the capacity of calcifying marine organisms to generate and maintain their skeletons. While many marine calcifying organisms precipitate low-Mg calcite or aragonite, the skeleton of echinoderms consists of more soluble Mg-calcite. To assess the impact of exposure to elevated temperature and increased pCO2 on the skeleton of echinoderms, in particular the mineralogy and microstructure, the starfish Aquilonastra yairi (Echinodermata: Asteroidea) was exposed for 90 days to simulated ocean warming (27 °C and 32 °C) and ocean acidification (455 µatm, 1052 µatm, 2066 µatm) conditions. The results indicate that temperature is the major factor controlling the skeletal Mg (Mg/Ca ratio and Mgnorm ratio), but not for skeletal Sr (Sr/Ca ratio and Srnorm ratio) and skeletal Ca (Canorm ratio) in A. yairi. Nevertheless, inter-individual variability in skeletal Sr and Ca ratios increased with higher temperature. Elevated pCO2 did not induce any statistically significant element alterations of the skeleton in all treatments over the incubation time, but increased pCO2 concentrations might possess an indirect effect on skeletal mineral ratio alteration. The influence of increased pCO2 was more relevant than that of increased temperature on skeletal microstructures. pCO2 as a sole stressor caused alterations on stereom structure and degradation on the skeletal structure of A. yairi, whereas temperature did not; however, skeletons exposed to elevated pCO2 and high temperature show a strongly altered skeleton structure compared to ambient temperature. These results indicate that ocean warming might exacerbate the skeletal maintaining mechanisms of the starfish in a high pCO2 environment and could potentially modify the morphology and functions of the starfish skeleton.

1. Introduction

The oceans are estimated to take up ~31% of the CO2 increase that is currently observed [1]. This is known to lead to lowered pH values of the seawater, resulting in a reduced carbonate saturation state (Ω), and changes in carbonate–bicarbonate ion balance, recognized as ocean acidification (OA) [2,3]. These changes in seawater chemistry lead to measurable reactions of marine species and ecosystems, and are known to have negative repercussions for many calcifying organisms [4,5,6,7], including certain scleractinian corals, bryozoans, molluscs, and echinoderms. Decreased carbonate ion concentration [CO32−] can disrupt the physiologically regulated biomineralization mechanism that generates, preserves and maintains calcium carbonate (CaCO3) structures, e.g., exoskeleton, test, spine, tube feet, teeth, pedicellariae and spicules [4,8,9,10]. This mechanism involves mineral formation, characteristics, morphogenesis, and organic molecules [9,11,12,13]. This is particularly the case for organisms precipitating a skeleton composed of high Mg-calcite (HMC) i.e., with a significant concentration of magnesium carbonate (MgCO3) in the carbonate (>4 mol% MgCO3) [14,15]. HMC is the most soluble of the polymorphs of crystalline CaCO3 and is thermodynamically metastable [14,16]. Furthermore, previous studies have shown that OA significantly affects the size and weight of shells or skeletons of many marine calcifiers [17,18,19]. In contrast, some calcifiers, mainly photosynthesizing or photosymbiotic ones, including some corals and algae, show positive responses in calcification and growth values as they benefit from increased CO2 concentrations by an enhanced photosynthesis rate, which provides additional potential energy for the calcification process [20].
At the same time, seawater temperature (ocean warming, OW) influences the ecophysiology of marine organisms [21], skeletal mineralogy [11,14,22,23,24,25,26,27], growth rate [28], and mineral growth control mechanisms [29]. Previous studies have found inconsistent reactions to increased temperature exposure, ranging from no effect to significant changes in skeletal mineralogy. For example, the skeletal Mg/Ca ratios of the scleractinian coral Acropora sp. [30], the sea urchin Paracentrotus lividus [25], the foraminifera Planoglabratella operculari, Quinqueloculina yabei [31] and Ammonia tepida [32] increase with temperature; conversely, the skeletal Mg/Ca ratios of the sea urchin Lytechinus variegatus was significantly lower in individuals kept at high temperature (~30 °C) compared to the ambient temperature (~26 °C) [26]. Skeletal Sr/Ca ratios decreases with increasing temperature in the scleractinian coral Acropora sp. [30,33], Sr/Ca ratios increases with increasing temperature in the foraminifera A. tepida [32] or Globigerinoides ruber [34], and is not significantly affected by temperature in the foraminifera Trifarina angulosa [35]. However, the effects of temperature on skeletal mineralogy and structure are still largely unclear and are likely influenced by phylogenetic factors, growth rate [22,23,36], latitude [24], biological ‘vital effects’ [37], and stage or species-specific differences [38].
The effects of combined OA and OW on marine calcifiers organisms are thought to be additive, antagonistic, or synergistic [39]. Previous meta-studies observed complex responses of calcifying marine organisms to combined OW and OA that vary from a significant negative effect to no effect on the organism [40]. Moreover, there is a trend toward enhanced sensitivity (i.e., the capability to sense and respond to environmental alterations) of biomineralization, growth, survival, and life stages development to OA in corals, echinoderm, and molluscs when being concurrently exposed to elevated temperatures [41]. Besides, there is growing evidence that elevated temperatures can exacerbate microstructure disruption caused by elevated pCO2 in ectotherm species, e.g., in some molluscs such as the giant clam Tridacna maxima [42] and the mussel Mytilus edulis [43,44], and echinoderms such as the sea urchin Tripneustes gratilla [45]. However, these synergistic effects of OA and OW seem to be complex, and the magnitude of effect sizes and organism response varies between taxa groups, trophic level, habitat and life stages [40,41,46].
Echinoderms comprise a wide variety of taxa, with a complex calcium carbonate biomineralization. Their skeleton is formed within the syncytium by progressive crystallization of a transient amorphous calcium carbonate phase (ACC) [47], through a biologically controlled intracellular mechanism within vesicles or vacuoles formed by fused cell membranes inside cells [11,22,48]. The echinoderm endoskeleton is composed of a complex three-dimensional porous microstructure (stereom) with connecting trabeculae (i.e., mesh-like interconnecting matrix rod of calcite skeleton) [11], which are composed of 99.8–99.9% weight/weight (w/w) HMC with Sr as the primary trace element [49,50], and the other 0.1–0.2% (w/w) of the skeleton being organic components consisting of proteins and glycoproteins, called the intrastereomic organic matrix (IOM) [51,52]. The IOM has critical functions in the biomineralization process during the transient ACC phase by stabilizing the skeleton, controlling mineral incorporation into the skeleton, and controlling the nucleation and morphology of the skeletal crystals [53,54]. The trace element concentration of biogenic CaCO3 is affected by biological factors, e.g., phylogeny, life stage, food supply, as well as by physical-chemical factors, e.g., temperature, salinity, seawater carbonate chemistry, concentration of Mg2+ and Ca2+ ions in the seawater, light, and hydrostatic pressure [14,27,37,49,55,56]. Seawater chemistry influences the mineralogy of the echinoderm skeleton by changing the physiological cost of sustaining the biological control of intracellular chemistry [57]. For echinoderms with their skeletons being composed of HMC (MgCO3 between 2.5% and 39% [8]), their skeletons become more soluble under OA conditions [16,50,58,59].
To gain a better understanding of the effects of combined OA and OW on biomineralization, systematic comparative studies across a phylogenetically diverse spectrum of taxa are needed [57]. The present study contributes to this goal by investigating the mineral composition and skeleton microstructure of Aquilonastra yairi (phylum Echinodermata, class Asteroidea, family Asterinidae) under controlled OA and OW conditions. This asterinid starfish thrives in the marine intertidal and coral reefs of the Red Sea and the Mediterranean Sea, where it lives in crevices and beneath corals or rocks [60]. It has an important ecological function as a grazer of marine algae, bacterial mats, detritus, and other fragments of food [61]. Previous studies have indicated deleterious effects of combined OA and OW on the physiological performances of asterinid starfish [62,63,64,65], while the effects of each stressor and their potential synergetic effects on skeletal mineral ratio and microstructure are currently still poorly understood.

2. Materials and Methods

2.1. Experimental Design and Control of Seawater Chemistry

In this study, starfish A. yairi was used as a model organism to investigate the combined effects of OW and OA. A total of 342 specimens of A. yairi (size 3–11 mm) from the cultivated stock of the MAREE (Marine Experimental Ecology facility) of ZMT, Bremen, Germany, were studied in the present experiment. Following acclimation procedures (see electronic Supplementary Materials), the starfish were cultured for 90-days in six different treatments, namely at two different temperatures (ambient temperature: 27 °C, and high temperature: 32 °C) crossed with three levels of pCO2 (low pCO2: 455 µatm, medium pCO2: 1052 µatm, and high pCO2: 2066 µatm). All six treatments were replicated in three aquaria (electronic Supplementary Materials Figure S1).
Target temperature and pCO2 levels were ramped up gradually over the first ten days to avoid physiological shock. Then the temperature (mean ± SE) in the treatment tanks was maintained at 27 ± 0.05 °C and 32 ± 0.08 °C, respectively, using a closed circle heating system (Heaters Titanium Tube 600 W, Schego Schemel & Goetz, Offenbach, Germany), controlled with a programmable thermostat. The mixture of the gas bubbled into the seawater in the bottom storage compartment sump was reached by blending compressed CO2-free air and compressed CO2 (pure CO2 provided by Linde GmbH, Pullach, Germany) using electronic solenoid-valve mass-flow controllers (HTK Hamburg GmbH, Hamburg, Germany) in accordance with the standard operating procedure (SOP) for ocean CO2 measurements [66]. Details of the seawater chemistry control and manipulation are provided in the electronic Supplementary Materials. The seawater parameters and carbonate chemistry for the experimental exposures are given in Table 1.

2.2. Skeletal Mineral Composition Analysis

On days 45 and 90 of the experimental treatment, six specimens from each replicate tank (i.e., 36 in total on each of those days) were randomly collected and rinsed in Milli-Q (18.2 MΩ) water before drying for ~48 h at 40 °C. In preparation for trace element analysis, to remove organic material from the skeletal matrix, dried starfish were soaked in hydrogen peroxide (H2O2) [67,68,69] for 24 h and subsequently cleaned mechanically, i.e., residual organic material was removed by forceps, and further potential contaminations were removed with deionized water in an ultrasonic bath. Then, the sample material was manually ground using a mortar and pestle. The powdered samples were kept at room temperature in sealed vials until analysis.
The element concentration of Ca, Mg and Sr in the skeleton was determined with a Spectro CIROS Vision (SPECTRO Analytical Instruments GmbH, Kleve, Germany) inductively coupled plasma optical emission spectroscope (ICP-OES). The samples (weighing 0.02–0.1 mg) were digested with concentrated nitric acid (HNO3) and H2O2 (high-purity of trace metal grade reagent). The solutions were then diluted to the acidity of 0.5 M HNO3 with aliquots of 0.1 mL and weighed again. Instrument calibration solutions (Inorganic VenturesTM 1000 ppm standard stock solution) were prepared using single-element standards in proportion to the A. yairi skeleton concentrations. Measurements of all starfish samples were done routinely against the international reference standard JLs−1, a coral in-house working standard (ZMT-CM1), and HNO3 blanks. Mg and Sr mineral elements are reported as a ratio over calcium (Ca), i.e., Mg/Ca, and Sr/Ca and over total skeletal material, i.e., Canorm, Mgnorm, and Srnorm, to account for minor organic material still present on or within the carbonate skeleton.

2.3. Analysis of the Skeleton Microstructure

One specimen from the 45-day and 90-day incubations from each treatment tank was randomly selected (n = 12), washed, and prepared for the SEM analysis. Each starfish was cut with dissecting scissors around the part of arms and cleaned of soft tissue. Organic material was dissolved using a 30% H2O2 solution buffered in NaOH (0.1 N) at room temperature for 24 h. Skeletons were then rinsed with distilled water repeatedly to remove any remaining organic material and then air-dried for 48 h at room temperature. Skeleton plates were then mounted on a stub with carbon-based tape and gold-sputtered (Cressington Sputter Coater 108 auto, Cressington Scientific Instruments, Watford, UK) for 30 s. Secondary electron images (SE) were generated with a scanning electron microscope (SEM; Tescan Vega3 XMU, Brno-Kohoutovice, Czech Republic) to characterize the skeleton microstructure, using a beam voltage of 5 kV for a magnification of up to 3000×. All SEM micrographs were examined for any visible differences between treatments, including signs of dissolution, surface smoothness, the shape of stereom pores, and the shape of inner matrix aperture pores.

2.4. Statistical Analysis

Statistical analysis was performed using the software R, version 4.1.3 [70]. Normality of data distribution and homogeneity of variance was tested with the Shapiro–Wilk statistic W test (α = 0.05) [71] and Levene’s test (α = 0.05) [72], respectively, and indicated that all data of skeletal mineral ratios were normally distributed and the homoscedasticity assumption for the data was equal. The effects of temperature, pCO2, incubation time and their interactions on skeletal mineral element to calcium ratios (Mg/Ca and Sr/Ca) and skeletal mineral element to total skeletal material ratios (Canorm, Mgnorm, and Srnorm) were examined using three-way analysis of variance (ANOVA), and Tukey HSD post hoc analyses were conducted using agricolae R-package 1.3-5 [73]. Temperature, pCO2, and incubation time were fixed factors, while skeletal mineral ratios were used as response variables. All statistics were evaluated with a significance level of α = 0.05.

3. Results

Over the duration of the experiment (90 days), the starfish mortality rate was low and only found in the high-temperature treatment. In general, the results indicate that elevated temperature and pCO2 changed the skeletal mineral composition, whereas elevated pCO2 affected skeletal microstructure in A. yairi.

3.1. Elemental Composition of Skeletal Carbonate

Overall, a relatively small range of Mg/Ca ratio values were observed across all our treatments (181.95–204.26 mmol/mol). The starfish had consistently higher Mg/Ca ratios in the 32 °C treatments (190.90 ± 1.41 mmol/mol, mean ± SE) than those held at 27 °C (187.59 ± 0.83 mmol/mol, mean ± SE) throughout all pCO2 concentration levels. Both incubation time and temperature had a main effect on skeletal Mg/Ca ratio (p = 0.049 and p = 0.033, respectively; Table 2). Inter-individual variability in skeletal Mg/Ca ratios was substantially higher in starfish subjected to high temperatures (32 °C) compared to those exposed to ambient temperatures (27 °C) in all pCO2 combined treatments (Figure 1A). No consistent pCO2 effect as the sole factor was found, and the Mg/Ca ratio displayed the typical parabolic responses to pCO2 (Figure 1A). Elevated pCO2 as the sole stressor did not significantly affect skeletal Mg/Ca ratios (p = 0.414, Table 2). The interaction of temperature: pCO2: incubation time on starfish skeletal Mg/Ca ratios was significant (p = 0.014, Table 2). However, Tukey’s HSD post hoc analysis did not reveal any significant interactions in Mg/Ca ratios (Table 2 and electronic Supplementary Materials Table S1).
Sr/Ca ratios ranged from 2.52 mmol/mol to 2.76 mmol/mol across treatments (Figure 1B). The Sr/Ca ratio at 32 °C had the highest fluctuation in values compared to 27 °C, where Sr/Ca ratios at 27 °C treatments (2.63 ± 0.01 mmol/mol, mean ± SE) were slightly higher than for the 32 °C treatments (2.62 ± 0.01 mmol/mol, mean ± SE) (Figure 1B). Inter-individual variability of Sr/Ca ratios were substantially higher for medium and high pCO2 treatments (1052 µatm, 2066 µatm) compared to low pCO2 (455 µatm) treatments; this was the case at both temperature levels (Figure 1B). No significant response of Sr/Ca ratio to differences in the pCO2 and temperature as combined or as sole stressors (Table 2). However, skeletal Sr/Ca ratios were significantly altered over incubation time (p = 0.006, Table 2), with increasing values from samples taken after 45 days compared to those collected after 90 days.
Canorm ratios showed relatively variable values, which ranged between 647.74 mg/g and 755.30 mg/g across all treatments (Figure 2A). Canorm ratios were changed over the incubation time (p = 0.036, Table 2). The Canorm ratios at 27 °C rose from 699.73 ± 5.41 mg/g (mean ± SE) at 45 days to 709.02 ± 4.33 mg/g (mean ± SE) at 90 days incubation time (enhanced 1.33%), whereas the Canorm ratios at 32 °C rose from 693.51 ± 8.93 mg/g (mean ± SE) at 45 days to 712.60 ± 7.48 mg/g (mean ± SE) at 90 days incubation time (enhanced 2.74%). Canorm ratios were not significantly affected by temperature (p = 0.838, Table 2) or pCO2 (p = 0.307, Table 2) as a single factor, nor as a combined factor (p = 0.250, Table 2).
Mgnorm ratios from the skeleton of A. yairi were significantly altered by incubation time (p = 0.001, Table 2). Mgnorm ratios increased from 45 days to 90 days in both temperature treatment conditions (Figure 2B). At 27 °C, the Mgnorm ratios increased from 109.57 ± 0.94 mg/g (mean ± SE) at 45 days to 112.98 ± 0.57 mg/g (mean ± SE) at 90 days incubation time (i.e., 3.11% increase), while at 32 °C, the Mgnorm ratios increased from 110.61 ± 1.04 mg/g (mean ± SE) at 45 days to 115.31 ± 0.63 mg/g (mean ± SE) at 90 days incubation time (4.25% increase). There was no significant effect of pCO2 nor any combined effect of the factors (Table 2). However, temperature led to a marginal increase in the Mgnorm ratios (p = 0.051, Table 2).
Srnorm ratios were altered over incubation time (p = 0.005, Table 2) at both temperature treatments (Figure 2B). The mean value of the Srnorm ratio at 27 °C increased by 2.38% from day 45 (3.36 ± 0.04 mg/g, mean ± SE) to day 90 (3.44 ± 0.02 mg/g, mean ± SD). Similarly, the mean value of Srnorm ratio at 32 °C increased from 45 days (mean ± SD, 3.30 ± 0.04 mg/g) to 90 days (3.47 ± 0.06 mg/g, mean ± SD) incubation time (5.15% increase). However, there was no significant effect of combined stressor factors nor solely stressor factors on Srnorm ratios (Table 2).

3.2. Skeletal Microstructure

High magnification SEM micrographs showed marked differences in skeletal structure between low pCO2 (455 µatm) compared to medium and high pCO2 treatments (1052 µatm, 2066 µatm, respectively) at both ambient and high temperatures (27 °C, 32 °C, respectively). The skeletal structure in low pCO2 crossed with ambient (27 °C: 455 µatm) and high temperatures (32 °C: 455 µatm) revealed no remarkable differences between 45-day and 90-day incubation time. The stereom pores were arranged equally in shape and the aperture pores of the inner matrix were relatively equal in shape, while the trabecular surface was smooth (Figure 3A,B,G,H and Table 3).
In contrast, after 45-day and 90-day incubation times, the skeleton from medium and high pCO2 treatments at ambient temperatures showed stereom structures that were more variable in shape compared to the control treatment, and signs of degradation, i.e., dissolution, were observed on the surface of the trabeculae (Figure 3C–F and Table 3). Furthermore, under high temperatures, these medium and high pCO2 treatments in addition result in signs of skeletal degradation observed at the trabeculae surface, while the apertures of the inner matrix pores were wider (i.e., un-equal in shape) compared to the control treatment and the ambient temperature crossed with medium and high pCO2 treatments (Figure 3I–L and Table 3).

4. Discussion

Our results imply that temperature plays a primary regulatory role in Mg concentration in the skeletal carbonate of A. yairi, where the Mg/Ca ratios increase under high-temperatures. This corroborates results from previous studies that found higher Mg concentrations associated with higher temperatures in echinoid and asteroid species [23,24,74]. This temperature association was previously attributed to kinetic factors affecting ion discrimination [23] and also to the physiological mechanisms that control Mg absorption in cells [75] during the biomineralization process. The increased Mg content of HMC was connected to an amorphous calcium magnesium carbonate (ACMC) precursor [76,77]. At high temperatures, the aqueous Mg2+ solvation energy barrier becomes lower [78]; hence this condition might favor more Mg2+ to be incorporated into the calcite lattice, encouraging the formation of ACMC, which later transforms into HMC.
Echinoderms are generally considered relatively poor regulators of internal acid–base balance, where the range of regulatory capacities is species-specific [79]. In hypercapnic conditions (high pCO2, low pH), echinoderms increase the bicarbonate concentration in their coelomic fluid and practice passive skeletal dissolution to support internal acid–base regulatory functions due to acidosis [80]. Higher Mg concentrations in the skeleton in conjunction with a degradation in the inner skeleton (see Section 3.2) due to skeletal dissolution, as documented in the current studies, support the assumption of a trade-off mechanism [65,81]. The released HMC mineral is then used as an active buffering mechanism to compensate for changes in internal pH that may help to avoid or reduce physiological impacts.
We noticed that no single (temperature or pCO2) nor combined stressor treatment affected skeletal Sr (Sr/Ca and Srnorm) in A. yairi. This contrasts with previous studies on the starfish Asterias rubens [49] and the sea urchin Paracentrotus lividus [25], which reported that the Sr/Ca ratio depended on temperature. These contrasting results indicate a species-specific skeletal Sr control mechanism in the echinoderm group that may respond to the stressors. The calcification rates might play an important role in skeletal Sr precipitation rather than direct dependence on temperature. It is difficult to discriminate the effects of temperature and pCO2 on skeletal Sr. Our data indicated that the skeletal Sr was increased over incubation time in all treatment combinations (electronic Supplementary Materials Figure S2) except the 27 °C: 455 µatm treatment, assuming that temperature and pCO2 might have an indirect effect on skeletal Sr through their control over the calcification process [23,34,69,82], which are common in biogenic calcites [83,84]. However, the incorporation pathways of Sr into the echinoderm ACC are still poorly understood, so further studies are needed.
We found a strong correlation between skeletal Sr and skeletal Mg (R2 = 0.04, p < 0.001, electronic Supplementary Materials Figure S3), as Srnorm increases with increasing Mgnorm ratio. Hence, Mg might play a role in affecting the Sr precipitation. Sr ions cannot easily substitute for Ca ions due to differences in cations ions size and weight (Sr2+ ionic radius of 1.18 Å: Ca2+ ionic radius of 1.00 Å [85]), which makes them incompatible in calcite [86]. High concentrations of Mg2+ (ionic radius of 0.72 Å [85]) that are incorporated in inorganic calcite can distort the crystal lattice (i.e., lattice deformation), which increases the size of calcium lattice positions and thus allows for increased incorporation of Sr2+ [87].
Similar to skeletal Sr, we found no direct influence of temperature or pCO2 on the Canorm ratio (Table 2). However, the highest inter-individual variability of Canorm indicates that temperature and/or pCO2 considerably alter the production of skeletal Ca in A. yairi, where Canorm ratios exhibited an increase with incubation time, except for the treatment of 27 °C: 455 µatm (~703 mg/g) where the ratio was relatively unchanged (electronic Supplementary Materials Figure S4). CaCO3 is more soluble at lower temperatures [88] and high pCO2 [20]. Under OW conditions, starfish seem to be able to boost their capacity to control calcification through the modulation of the intracellular calcifying fluids pH to produce CaCO3. This might provide them with higher resistance and resilience towards the effects of OA. The increased energetic costs of this physiological response are indicated by elevated respiration rates [65]. Hence, in the long term, this phenotypic plasticity and biocalcification control mechanism might have further physiological implications for the starfish.
The hypercapnic conditions of the high pCO2 treatment in our experiment have resulted in visible adverse effects on the skeletal structures in both ambient as well as elevated temperatures (Figure 3, Table 3). We detected changes in the stereom pores, the inner matrix pores and degradation of the skeleton surface through reduced deposition or dissolution, which increased over incubation time in the starfish that were exposed to medium and high pCO2. These skeletal alterations might be due to the thin layer of epithelial tissue covering the starfish body wall [89], which acts as a protective membrane between the skeleton and the seawater [90]. Furthermore, the degradation of the epithelial tissue could also explain why the combination of OW with OA enhances the degradation of the skeletal structure that was observed in the high pCO2 treatment (32 °C: 2066 µatm). We hypothesize that the epithelial tissue might become weaker in its function due to degradation of the epithelium cells during long-term experiments. Epithelial tightness is controlled by a protein series that molds a seal among epithelial cells [90], which are sensitive to elevated temperature.
The seawater chemistry factor, which was underlying the altered skeletal structure in A. yairi might involve calcium carbonate saturation state (Ω). Skeletal degradation provides evidence that the skeleton of A. yairi was negatively affected by lowered calcite saturation state (Ωca) as a function of increasing pCO2 (Table 1), which directly affects the biomineralization and dissolution of the CaCO3 in skeletal structures [17,91]. Near Ωca ~3, we found stereom alteration and degradation signs in trabeculae, which indicates that A. yairi was facing difficulty in producing and maintaining their skeleton compared to low pCO2 treatments (Ωca > 5). Previous studies found that the skeletal morphometric development of echinoid (e.g., sea urchin Lytechinus variegatus) was significantly affected by Ωca 2.53 [92]. Furthermore, increased susceptibility of the skeleton to dissolution through OA was suggested to be due to the skeleton composed of HMC, which is 30 times more soluble than pure calcite [47,93]. When the qualitative approach depicts changes in the skeleton micro-morphology because of exposure to elevated pCO2 and temperature (Table 3, Figure 3), further investigation using quantitative analysis (e.g., stereom and inner matrix pore size) is required to quantify the alteration magnitude in skeleton microstructure as the deleterious impact of OA and OW [94,95].
Under OW and OA scenarios, calcifying marine organisms are expected to face unfavorable conditions to produce and maintain their skeletal HMC in order to sustain their biomechanical functions. Phenotypic plasticity requires re-allocation of energy as a trade-off and may represent a potential key mechanism for species viability [96]. For A. yairi, this mechanism might reflect strategies to maintain their skeletal HMC in a low pH environment by modulating their cellular chemistry to create isolated microenvironments of deposition, producing the specific mineral they necessitate, which involves significant energy re-allocation [97]. Since the asterinid starfish produces HMC, which is more soluble than calcite and aragonite, it signifies that more energy will be required to preserve the calcification process as the mechanism in both constructing and maintaining their skeletal components, leading to energy trade-offs against other physiological processes [65,98].
Changes in the shape of the stereom pores and degradation of the starfish skeleton potentially have implications for skeletal strength, stiffness, and function. Weakening of the structure possibly will reduce locomotion performance and result in a lower ability to resist predators and to face ocean currents, which then conveys consequences to the benthic community structure.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/jmse10081065/s1; Table S1: Tukey HSD post hoc test for the interactive effects of incubation time (45 and 90 days), pCO2 (455 µatm, 1052 µatm, 2066 µatm) and temperature (27 °C, 32 °C) on skeletal Mg/Ca ratio of A. yairi using the ‘agricolae R-package’ for multiple comparisons to interrogate the main effects of incubation time, temperature and pCO2 (incubation time: pCO2: temperature = 0.014, Table 2); Figure S1: Schematic of ocean acidification and ocean warming experimental design with a fully factorial combination of low pCO2 (455 μatm), moderate pCO2 (1052 μatm), and high pCO2 (2066 μatm) treatments with ambient temperature (27 °C) and high temperature (32 °C) treatments; Figure S2: Skeletal Sr values over incubation time in A. yairi exposed to elevated temperatures levels (27 °C and 32 °C) crossed with increased pCO2 concentrations (455 µatm, 1052 µatm, and 2066 µatm). (A) Sr/Ca ratio (mmol/mol), (B) Srnorm ratio (mg/g). Data were presented as mean (n = 36); Figure S3: Correlation of Srnorm ratios (mg/g) against Mgnorm ratios (mg/g) (R2 = 0.44, F1, 34 = 26.78, p = 1.019 × 10−5) in A. yairi exposed to elevated temperatures levels (27 °C and 32 °C) crossed with increased pCO2 concentrations (455 µatm, 1052 µatm, and 2066 µatm). (n = 36); Figure S4: Skeletal Canorm (mg/g) ratio over incubation time in A. yairi exposed to elevated temperatures levels (27 °C and 32 °C) crossed with increased pCO2 concentrations (455 µatm, 1052 µatm, and 2066 µatm). Data were presented as mean (n = 36). For more details, please see [66, 99,100,101,102,103,104,105,106].

Author Contributions

Conceptualized and designed: M.K. and H.W.; methodology: M.K., S.S.D., M.S. and H.W.; investigation: M.K.; resources: M.K. and H.W.; sample processing and analysis: M.K.; data curation: M.K.; writing original draft preparation: M.K.; visualization: M.K.; writing-review and editing: M.K., S.S.D., M.S. and H.W.; supervision: H.W. All authors have read and agreed to the published version of the manuscript.

Funding

This research project was supported by the Academy Doctoral Research—Grant Leibniz Centre for Tropical Marine Research (ZMT), Germany and the Ministry of Education, Culture, Research, and Technology (MoECRT), Republic of Indonesia—Asian Development Bank (ADB) AKSI Project [grant number L3749-INO] to M.K.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The datasets used and analyzed during the current study are present in the manuscript and/or supplementary materials. Data are available upon request from the corresponding author.

Acknowledgments

We would like to thank Andreas Kunzmann (ZMT, Bremen, Germany) for his advice on the starfish A. yairi as a model organism. Many thanks are due to the ZMT laboratory staff, namely Jule Mawick, Sebastian Flotow, Silvia Hardenberg, José Garcia, Nico Steinel, Matthias Birkicht and Fabian Hüge.

Conflicts of Interest

On behalf of all authors, the corresponding author states that there is no conflict of interest.

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Jmse 10 01065 g001 550
Figure 1. Changes in skeletal properties (A) Mg/Ca ratios (mmol/mol) and (B) Sr/Ca ratios (mmol/mol) of skeletal carbonate in A. yairi exposed to different temperatures (27 °C and 32 °C) crossed with different pCO2 concentrations (455 µatm, 1052 µatm, and 2066 µatm) measured after 45 and 90 days of incubation (n = 36). Dots represent individual skeletal mineral values, displayed with jitter to avoid overlap.
Figure 1. Changes in skeletal properties (A) Mg/Ca ratios (mmol/mol) and (B) Sr/Ca ratios (mmol/mol) of skeletal carbonate in A. yairi exposed to different temperatures (27 °C and 32 °C) crossed with different pCO2 concentrations (455 µatm, 1052 µatm, and 2066 µatm) measured after 45 and 90 days of incubation (n = 36). Dots represent individual skeletal mineral values, displayed with jitter to avoid overlap.
Jmse 10 01065 g001
Jmse 10 01065 g002 550
Figure 2. Ratios between skeletal mineral element to total skeletal material (A) Canorm ratios (mg/g), (B) Mgnorm ratios (mg/g), and (C) Srnorm ratios (mg/g) in A. yairi exposed to elevated temperatures levels (27 °C and 32 °C) crossed with increased pCO2 concentrations (455 µatm, 1052 µatm and 2066 µatm) measured after 45 and 90 days of incubation (n = 36). Dots represent individual skeletal mineral values, displayed with jitter to avoid overlap.
Figure 2. Ratios between skeletal mineral element to total skeletal material (A) Canorm ratios (mg/g), (B) Mgnorm ratios (mg/g), and (C) Srnorm ratios (mg/g) in A. yairi exposed to elevated temperatures levels (27 °C and 32 °C) crossed with increased pCO2 concentrations (455 µatm, 1052 µatm and 2066 µatm) measured after 45 and 90 days of incubation (n = 36). Dots represent individual skeletal mineral values, displayed with jitter to avoid overlap.
Jmse 10 01065 g002
Jmse 10 01065 g003a 550Jmse 10 01065 g003b 550Jmse 10 01065 g003c 550
Figure 3. SEM micrographs of the skeletal microstructure of A. yairi cultured under two temperature levels (27 °C and 32 °C) crossed with three pCO2 concentrations (455 µatm, 1052 µatm and 2066 µatm). Stereom microstructure after 45 (A,C,E,G,I,K) and 90 days (B,D,F,H,J,L) of incubation time. sp: stereom pore; st: skeleton trabeculae; imp: stereom inner matrix pore; ⓐ the galleries stereom pores are less-equal in shape; ⓑ dissolution in calcium carbonate skeleton; ⓒ increased size of inner matrix aperture pores. Technical image acquisition, SEM mode: SE, SEM HV: 5.0 kV, SEM magnification: 3000×.
Figure 3. SEM micrographs of the skeletal microstructure of A. yairi cultured under two temperature levels (27 °C and 32 °C) crossed with three pCO2 concentrations (455 µatm, 1052 µatm and 2066 µatm). Stereom microstructure after 45 (A,C,E,G,I,K) and 90 days (B,D,F,H,J,L) of incubation time. sp: stereom pore; st: skeleton trabeculae; imp: stereom inner matrix pore; ⓐ the galleries stereom pores are less-equal in shape; ⓑ dissolution in calcium carbonate skeleton; ⓒ increased size of inner matrix aperture pores. Technical image acquisition, SEM mode: SE, SEM HV: 5.0 kV, SEM magnification: 3000×.
Jmse 10 01065 g003aJmse 10 01065 g003bJmse 10 01065 g003c
Table
Table 1. Seawater chemistry values measured during a 90-day experimental period for A. yairi reared under two temperature levels (27 °C and 32 °C) crossed with three levels of pCO2 (455 µatm, 1052 µatm, and 2066 µatm). AT, total alkalinity; DIC, dissolved inorganic carbon; pCO2, partial pressure of CO2; [CO32−], carbonate ion concentration; [HCO3−], bicarbonate ion concentration; [CO2], dissolved CO2; ΩCa, calcite saturation state; ΩAr, aragonite saturation state. Data are presented as mean values ± SE.
Table 1. Seawater chemistry values measured during a 90-day experimental period for A. yairi reared under two temperature levels (27 °C and 32 °C) crossed with three levels of pCO2 (455 µatm, 1052 µatm, and 2066 µatm). AT, total alkalinity; DIC, dissolved inorganic carbon; pCO2, partial pressure of CO2; [CO32−], carbonate ion concentration; [HCO3−], bicarbonate ion concentration; [CO2], dissolved CO2; ΩCa, calcite saturation state; ΩAr, aragonite saturation state. Data are presented as mean values ± SE.
TreatmentMeasured Parameters
Salinity (PSU)Temperature (°C)pH(NBS scale)pH(total scale)AT
(µmol/kg-SW)		DIC
(µmol/kg-SW
27 °C: 455 µatm34.56 ± 0.1227.48 ± 0.068.13 ± 0.008.00 ± 0.002504.42 ± 15.332168.86 ± 15.23
27 °C: 1052 µatm34.73 ± 0.0627.23 ± 0.047.87 ± 0.017.74 ± 0.012514.45 ± 16.782340.99 ± 11.62
27 °C: 2066 µatm34.75 ± 0.0527.34 ± 0.037.60 ± 0.017.47 ± 0.012539.83 ± 38.862479.40 ± 36.58
32 °C: 455 µatm34.65 ± 0.0832.03 ± 0.058.13 ± 0.008.00 ± 0.002510.27 ± 47.182134.38 ± 38.40
32 °C: 1052 µatm34.78 ± 0.0532.10 ± 0.047.87 ± 0.007.74 ± 0.002532.19 ± 40.052325.20 ± 40.02
32 °C: 2066 µatm34.76 ± 0.0232.20 ± 0.087.60 ± 0.017.47 ± 0.012584.38 ± 41.932493.30 ± 37.87
TreatmentCalculated Parameters
pCO2 (µatm)[CO32−] (µmol/kg-SW)[HCO3−] (µmol/kg-SW)[CO2] (µmol/kg-SW)ΩCaΩAr
27 °C: 455 µatm456.13 ± 8.24245.64 ± 2.521911.05 ± 14.9412.17 ± 0.225.96 ± 0.063.96 ± 0.04
27 °C: 1052 µatm1059.58 ± 32.04138.12 ± 4.642178.74 ± 9.5828.42 ± 0.863.35 ± 0.112.22 ± 0.07
27 °C: 2066 µatm2075.40 ± 30.9981.18 ± 2.352342.71 ± 34.2955.51 ± 0.821.97 ± 0.061.31 ± 0.04
32 °C: 455 µatm453.78 ± 6.51273.82 ± 8.231849.66 ± 30.7710.90 ± 0.166.71 ± 0.204.52 ± 0.14
32 °C: 1052 µatm1045.15 ± 44.00162.68 ± 4.672147.47 ± 38.5925.05 ± 1.063.98 ± 0.112.69 ± 0.08
32 °C: 2066 µatm2057.31 ± 74.4299.46 ± 4.642344.63 ± 34.9449.20 ± 1.782.44 ± 0.111.64 ± 0.08
Table
Table 2. Summary of three-way ANOVA results for the skeletal mineral ratios of A. yairi exposed to temperature (27 °C, 32 °C) crossed with elevated pCO2 (455 µatm, 1052 µatm, 2066 µatm) treatments for 45 and 90 days incubation time. Tukey HSD post hoc tests were performed where ANOVA results indicated significant effects of one or several factors (incubation time, pCO2, temperature) with p-values adjusted for multiple testing (padj). Bold terms indicate a significant difference (p < 0.05).
Table 2. Summary of three-way ANOVA results for the skeletal mineral ratios of A. yairi exposed to temperature (27 °C, 32 °C) crossed with elevated pCO2 (455 µatm, 1052 µatm, 2066 µatm) treatments for 45 and 90 days incubation time. Tukey HSD post hoc tests were performed where ANOVA results indicated significant effects of one or several factors (incubation time, pCO2, temperature) with p-values adjusted for multiple testing (padj). Bold terms indicate a significant difference (p < 0.05).
Skeletal Mineral RatiodfFPr (<F)Post-Hoc Test Result
Mg/Ca ratio
Incubation time14.3190.04990 days > 45 days
pCO220.9150.414
Temperature15.1440.03332 °C > 27 °C
Incubation time: pCO220.7150.500
Incubation time:temperature10.0390.845
pCO2: temperature20.4780.626
Incubation time: pCO2: temperature25.1430.014n.s., electronic Supplementary Materials Table S1
Residuals24
Sr/Ca ratio
Incubation time19.0270.00690 days > 45 days
pCO220.4050.671
Temperature10.4810.495
Incubation time: pCO221.7940.188
Incubation time: temperature11.5190.230
pCO2: temperature22.0160.155
Incubation time: pCO2: temperature21.1320.339
Residuals24
Canorm ratio
Incubation time14.9510.03690 days > 45 days
pCO221.2420.307
Temperature10.0430.838
Incubation time: pCO221.7110.202
Incubation time: temperature10.590.450
pCO2: temperature21.4710.250
Incubation time: pCO2: temperature21.650.213
Residuals24
Mgnorm ratio
Incubation time124.5230.00190 days > 45 days
pCO220.5820.566
Temperature14.2060.05132 °C > 27 °C
Incubation time: pCO220.6160.548
Incubation time: temperature10.6210.438
pCO2: temperature20.6090.552
Incubation time: pCO2: temperature22.1330.140
Residuals24
Srnorm ratio
Incubation time19.8140.00590 days > 45 days
pCO220.5000.613
Temperature10.1560.696
Incubation time: pCO221.8620.177
Incubation time: temperature11.4050.248
pCO2: temperature21.6550.212
Incubation time: pCO2: temperature20.7070.503
Residuals24
Table
Table 3. Skeletal microstructure characteristics of A. yairi under crossed temperatures and pCO2 conditions at different incubation times as observed with scanning electron microscopy (SEM). ND: the skeleton surface is smooth and has no signs of degradation; DS: the skeleton surface had degradation signs; HD: the skeleton surface had high degradation signs; ES: the stereom pores were equal in shape; US: the stereom pores were un-equal in shape; HU: the stereom pores were highly un-equal in shape; EP: the inner matrix aperture pores were relatively equal in shape; UP: the inner matrix aperture pores were relatively un-equal in shape (wider). (n = 12).
Table 3. Skeletal microstructure characteristics of A. yairi under crossed temperatures and pCO2 conditions at different incubation times as observed with scanning electron microscopy (SEM). ND: the skeleton surface is smooth and has no signs of degradation; DS: the skeleton surface had degradation signs; HD: the skeleton surface had high degradation signs; ES: the stereom pores were equal in shape; US: the stereom pores were un-equal in shape; HU: the stereom pores were highly un-equal in shape; EP: the inner matrix aperture pores were relatively equal in shape; UP: the inner matrix aperture pores were relatively un-equal in shape (wider). (n = 12).
Incubation TimeTemperaturepCO2Skeletal SurfaceStereom PoresInner Matrix Pores
45 days27 °C455 µatmNDESEP
1052 µatmDSUSEP
2066 µatmDSUSUP
32 °C455 µatmNDESEP
1052 µatmDSUSUP
2066 µatmHDHUUP
90 days27 °C455 µatmNDESEP
1052 µatmDSUSEP
2066 µatmDSUSUP
32 °C455 µatmNDESEP
1052 µatmDSUSUP
2066 µatmHDHUUP
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Khalil, M.; Doo, S.S.; Stuhr, M.; Westphal, H. Ocean Warming Amplifies the Effects of Ocean Acidification on Skeletal Mineralogy and Microstructure in the Asterinid Starfish Aquilonastra yairi. J. Mar. Sci. Eng. 2022, 10, 1065. https://doi.org/10.3390/jmse10081065

AMA Style

Khalil M, Doo SS, Stuhr M, Westphal H. Ocean Warming Amplifies the Effects of Ocean Acidification on Skeletal Mineralogy and Microstructure in the Asterinid Starfish Aquilonastra yairi. Journal of Marine Science and Engineering. 2022; 10(8):1065. https://doi.org/10.3390/jmse10081065

Chicago/Turabian Style

Khalil, Munawar, Steve S. Doo, Marleen Stuhr, and Hildegard Westphal. 2022. "Ocean Warming Amplifies the Effects of Ocean Acidification on Skeletal Mineralogy and Microstructure in the Asterinid Starfish Aquilonastra yairi" Journal of Marine Science and Engineering 10, no. 8: 1065. https://doi.org/10.3390/jmse10081065

APA Style

Khalil, M., Doo, S. S., Stuhr, M., & Westphal, H. (2022). Ocean Warming Amplifies the Effects of Ocean Acidification on Skeletal Mineralogy and Microstructure in the Asterinid Starfish Aquilonastra yairi. Journal of Marine Science and Engineering, 10(8), 1065. https://doi.org/10.3390/jmse10081065

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