Weakening Mechanisms of the Serpulid Tube in a ... - ACS Publications

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Weakening Mechanisms of the Serpulid Tube in a High-CO2 World Chaoyi Li,† Vera B. S. Chan,† Chong He,‡ Yuan Meng,† Haimin Yao,‡ Kaimin Shih,§ and Vengatesen Thiyagarajan*,† †

The Swire Institute of Marine Science and School of Biological Sciences, The University of Hong Kong, Pokfulam, Hong Kong SAR Department of Mechanical Engineering, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong SAR § Department of Civil Engineering, The University of Hong Kong, Pokfulam, Hong Kong SAR ‡

S Supporting Information *

ABSTRACT: Many benthic marine organisms produce calcium carbonate (CaCO3) structures for mechanical protection through a biologically controlled calcification process. However, the oceans are becoming unfavorable for calcification because of the stress associated with ocean acidification (OA) and associated chemical changes such as declining saturation state of CaCO3 and decreasing seawater pH. This work studies the impacts of OA-driven decreased pH on the calcareous tubes produced by the serpulid tubeworm Hydroides elegans. Tubes grown under control and OA experimental conditions were measured for structural and mechanical properties, and their mechanical properties were further interpreted using finite element analysis (FEA). The near-future predicted pH value of 7.8 altered tube ultrastructure, volume, and density and decreased the mean tube hardness and elasticity by ∼80 and ∼70%, respectively. The crushing force required for breaking the tube was reduced by 64%. The FEA results demonstrated how a simulated predator attack may affect the structure with different structural and mechanical properties and consequently shift the stress development and distribution in the tubes, causing a more concentrated stress distribution and therefore leading to a lower ability to withstand attacks.

1. INTRODUCTION Coastal oceans are frequently more acidic than open oceans due to anthropogenic nutrient enrichment and upwelling.1 Consequently, the pH variability is greater in coastal areas than in open oceans.2 Within this century, rising levels of anthropogenic CO2 are expected to cause a reduction in the pH and undersaturation of CaCO3 minerals in coastal waters. These seawater chemistry changes/shifts are referred to as “ocean acidification” (OA).3,4 While mobile species may be expected to migrate away from their current habitat in the localized acidified area,5,6 a recent meta-analysis confirmed that the majority of calcifying benthic marine species does not have the necessary acclimatory or local adaptive capacity to cope with the rapidly decreasing pH due to OA.7 There have been concerted efforts to examine the sensitivity of calcifying marine organisms, such as corals,8,9 clams,10 oysters,11,12 sea urchins,13 and tubeworms.14 Although recent studies have advanced our understanding of OA and its impacts on calcifying organisms, much of the published research has focused on physiology, calcification rate, survival, and growth.15,16 Calcification responses commonly include growth rate, overall calcification rate, microstructure observation, and mechanical tests.17−21 Furthermore, marine species are often found to produce weaker shells under OA. As the CaCO3 saturation state drops with decreasing pH or increasing OA © XXXX American Chemical Society

stress, calcifying animals may lose the ability to produce ordered biomineral morphology, and this may pose mechanical challenges to their shells.14,22 Similar to many other marine organisms that calcify with a controlled mechanism,23−25 serpulid tubeworms have calcareous tubes that protect them from predators and environmental stressors.26,27 The metamorphosis of serpulid worms on benthic substrate is immediately followed by the rapid production of calcareous tubes. This rapid calcification process is an ideal model system for OA studies.28 Besides having this unique calcification process, our study species, Hydroides elegans, is ecologically and economically important due to its reef building and biofouling ability, especially in warmer waters.29 Although the ultrastructures and the evolution of tube mineralogy in serpulid have been quite well studied,27,30,31 our understanding of the effects of OA on tube structure is still limited. At the near-future projected OA level at decreased pH 7.8, H. elegans can produce their tubes, but they produce them at a slower rate.32 Although OA impacts on tube mineral composition, ultrastructure, and mechanical properties have Received: April 4, 2014 Revised: September 17, 2014 Accepted: November 9, 2014

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dx.doi.org/10.1021/es501638h | Environ. Sci. Technol. XXXX, XXX, XXX−XXX

Environmental Science & Technology

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been examined in this species,14 the mechanisms through which OA alters the tube mechanical properties and its capacity to withstand a predatory attack are yet to be studied. In fact, the effectiveness of calcareous structure in terms of providing mechanical protection and structural support can be evaluated by a computational method known as finite-element analysis (FEA).33,34 In addition, multiple nanoindentations and microcomputed tomography (microCT) methods provide good resolution and are routinely used in materials science research35,36 including many biomaterial studies.37,38 Multiple nanoindentation can quantitatively map and measure the heterogeneity of the mechanical strength along the depth of the tube and results from structural or chemical composition anomalies.39 The size and mechanical parameters can be further analyzed by FEA with an engineering mechanical perspective to study the mechanical reaction of skeletons to external loading due to predator attack.39−41 In the complex and diverse world of marine invertebrate biomineralization, FEA can potentially unify the examination of mechanical behavior in different calcified products; such information can improve predictive understanding at the community and ecosystem levels.42 To gain insight into the ways in which calcareous structures built by a marine organism respond to the elevated CO2 driven pH reduction (OA), we examined changes in the ultrastructure and mechanical properties of tubes built by tubeworm H. elegans at pH values representing contemporary (pH = 8.1) and near-future projected scenarios (pH =7.8). Size parameters and structural features were evaluated by SEM and microCT. Mechanical properties were analyzed using a more detailed approach with multiple-nanoindentations and a more holistic approach with crushing tests. The information about size and mechanical parameters were further measured for FEA. The results obtained from FEA provided both qualitative and quantitative insights into the stress distribution and development within the tubes under OA.

century to pH 7.8 due to ocean acidification.46 Thus, the selected experimental decreased pH of 7.8 represents both the current extreme levels as well as the future average. The embryos were raised to trochophore larvae in 5 L culture tanks and were grown to competent larvae. Plastic plates with 7-day-old natural biofilms were deployed in each container to induce larval metamorphosis.43 Competent larvae readily attached to the plates and metamorphosed into juveniles that developed further to day 18 after metamorphosis using standard culture procedures.43,47 Once every 2−3 days, the culture seawater was replenished, and animals were fed with Isochrysis galbana, algal food ad libitum (about 105 cells ml−1). The pH levels were maintained by bubbling with ambient air (for control pH 8.1) or the CO2-enriched air (for decreased pH 7.8) in the respective culture tanks. There were four replicate culture tanks in each treatment group. The CO2-enriched air was prepared using a mixture of air/CO2 and regulated by a flow meter. The CO2 concentration in the inflow air was frequently measured using a Quantek Model 906 Carbon Dioxide Analyzer (Quantek Instruments, Inc., Grafton, MA). Seawater pH (NBS scale), salinity, temperature, and total alkalinity values were monitored daily by a pH meter (SG2, Metelo-Toledo, Hong Kong), a refractometer, a thermometer, and an Alkalinity Titrator (Apollo SciTech, Inc., Bogart, GA) respectively. These values were subsequently used to calculate the entire carbonate system variables using the standard protocol.48 After the experimental period, tubeworms from all the eight culture tanks were washed with Milli-Q water to remove salts and were killed with 70% ethanol, which is know to maintain any unstable amorphous calcium carbonate (ACC).49 Empty tubes were air-dried and kept at room temperature until the ultrastructure and mechanical properties analyses. 2.2. Tube Ultrastructure, Thickness, and Radius Measurement. Tubes were dislodged from the substrate, embedded, and sectioned to identify the ultrastructures using scanning electron microscopy (SEM).50 Resin embedded tube specimens were manually sectioned perpendicular to the longitudinal axis, excess resin surrounding the tube section was trimmed away. The sectional surfaces were cut again with the control of an ultramicrotome (Ultracut S, Leica, Germany) using a glass knife and finally a diamond knife (DiATOME Ultra 45°) to provide a smooth polished surface. Sectioned surfaces were etched for 1 min with 0.5 M EDTA. Prior to scanning electron microscopy, tubes were mounted on aluminum stubs using carbon adhesive tape with the sectioned side facing up. The surrounding vertical resin surfaces were covered with silver to minimize charging, and then the specimen was sputter coated with a 50 nm thick gold− palladium alloy. Tube ultrastructure was visualized at an accelerating voltage of 5 kV, and the distribution of Sr and Mg in the CaCO3 minerals was determined using elemental analysis at an accelerating voltage of 20 kV by energy-dispersive X-ray spectroscopy (SEM-EDS) using a LEO 1530 Gemini FSEM (Zeiss, Germany). The average thickness and circular radius were obtained from each image using imageJ (ver 1.46r, NIH). 2.3. Tube density, Volume, and 3D Surface Scan. Nondestructive three-dimensional digital information relating to the surface topography and physical properties were obtained using microcomputed tomography (micro-CT) scanning system (SkyScan 1076, Skyscan, Belgium) with a 3 × 10−6 cubic mm voxel size with spatial resolution of 15 μm.

2. MATERIALS AND METHODS 2.1. Study Organism, pH Perturbation Experiment, and Sample Collection. The serpulid polychaete tubeworm H. elegans is a tube-forming species that can form calcareous reefs. We used the tubeworm as the experimental model because (1) it is commercially important as a biofouling organism,28 (2) it can be cultured year-round in the laboratory,43 (3) it produces a bimineralic layered tube,14 and (4) it is a sensitive model species that can be used to help us understand ocean acidification impacts.32 Mature tubeworms were obtained from a fish farm in Yung Shue O (22°27′ N, 114°23′ W) in March 2011, located in relatively sheltered coastal waters in Hong Kong. From winter to spring, the eastern waters have an average seawater salinity of ∼33 ‰, a temperature range of ∼18−24 °C, and a pH of ∼8.1. Eggs and sperms from multiple parental individuals (>100) were mixed to obtain a homogeneous embryo population for a CO2 perturbation experiment described as follows.44 The effects of CO2-driven reduction in ocean pH (ocean acidification) on the tube ultrastructure and mechanical properties were compared at ambient pH 8.1 (control) and decreased pH 7.8 (treatment). The decreased pH 7.8 treatment level was selected based on two criteria: (1) the average late spring or early summer pH in Hong Kong coastal waters naturally varies between pH 8.2 and 7.8 due to summer monsoon precipitation,45 and (2) the average current seawater pH of 8.1 is projected to decrease by 0.3 units within this B



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were assumed fixed on the substrate as preset boundary condition. Materials were assumed purely elastic with Young’s modulus calculated from the reduced modulus assuming that Poisson’s ratio is 0.3. To capture the variation of mechanical properties along the thickness of sample produced from treatment condition, the Young’s modulus resulted from the control and treatment conditions were applied for analysis (Supplementary Table 2, Supporting Information). A plain strain element of CPS8 was used in our analysis. A rigid flat plane was compressed onto the shell to simulate the external loading from predator attack as Common predators’ (e.g., flatworms, sea urchins, crabs, fishes and various molluscs55) weapons are much larger that the tube of tubeworm. A plane force is also consistent with our results obtained from microforce testing when the tubes were crushed by a plane. 2.7. Data Analysis. The effect of decreased pH seawater on tube thickness, circular radius, tube volume, tube density, hardness, Young’s modulus, and breaking force were statistically compared using Student’s t test. F-test for variance homogeneity suggested the variances were homogeneous or not. Student’s t test for equal variance was applied when p > 0.05, while t test for unequal variance was applied when p < 0.05.

With this resolution, the main structures of the tube can be captured for measurement of tube volume and density. Each plastic substrate, with multiple tube specimens (4−8 individuals), was placed in a falcon tube and held securely in the micro-CT chamber for scanning. To clearly distinguish the boundary of each tubeworm for the calculation of tube volume and density, we avoided colonies and tested a few individually settled tubeworms. The acrylic plastic substrates demonstrated a much weaker signal than the calcium carbonate tubes, and were considered as a removable background signal. The 3D digital data were converted from 98 to 263 images using reconstruction software CTvol v 2.2.1.0 (SkyScan, Kontich, Belgium) for the tube volume and density of 1−2 tube individuals in each replicate plate. A universal signal threshold setting was applied to measure the tube volume and density. Tube volume was measured and calculated from the number of pixels occupied by each tube; the density measurement was a relative measurement as standardized using phantoms used for bone density measurement.51 The average values of volume and density of tubes in each replicate plate were obtained and analyzed for the effects of seawater pH treatment. 2.4. Multiple Nanoindentation Analysis. After SEM imaging, tube samples were polished again by controlled sectioning with a diamond knife using an ultramicrotome (Ultracut S, Leica), as necessary for the nanoindentation analysis to avoid roughness and topography from interfering the indentation at the nano scale.52 Nanoindentations were performed by using Berkovich tip (TI 900, Hysitron, MN, USA).53 Multiple nanoindentations were carried out along the thickness of the shell, from the exterior side (denoted as normalized depth of 0) to the lumen side (denoted as normalized depth of 1), using an 8−10 indent-per-row pattern and a peak load of 10 mN. A map of mechanical properties (hardness and reduced modulus) as a function of normalized depth (values of 0−1) was obtained after collecting enough indentation results from samples tested (n = 6−9). Each data point on the map stands for an individual indent from a tubeworm tested, and the final map is a collection plot of all data collected. The hardness and modulus from each indentation were reduced from the loading−unloading curve by using the Oliver−Pharr model.54 2.5. Crushing Force Measurement. The overall mechanical strength of shells can be characterized by the crushing force, which is the maximum sustainable force under compression. The crushing force was measured using the microforce testing system (Tytron 250, MTS System Co., Eden Prairie, MN). In each set of measurement, a substrate plate was secured by a mechanical clamp of the testing apparatus. A metal plane (diameter of 1.5 cm) was used to test the mechanical response of the tubes on the plate. Individual tested tubes generated a displacement−force curve to accurately measure the crushing force. The displacement control load was applied with speed of 0.05 mm/sec. Based on the obtained force− displacement curve, the crushing force was taken as the corresponding force at the “yielding” point on the curve. For each treatment, 3−5 tubes per replicate were analyzed. 2.6. Finite Element Modeling. Finite element analysis (Abaqus, Dassault Systems, France) was conducted to understand the mechanical behavior of tubes subjected to the treatment and control conditions. The exoskeleton was modeled as a semicylindrical shell with cross-sectional dimension obtained from the measurements. Because the tubes were attached to the substrate, the two ends of the shell

3. RESULTS The control seawater at average pH of 8.21 ± 0.06 was maintained through bubbling of ambient air, whereas the increased concentration of CO2 reduced the seawater pH to 7.84 ± 0.04. The aragonite saturation state (Supplementary Table 1, Supporting Information) decreased significantly from 3.3 in the control to 1.69 in the decreased pH treatment. The total alkalinity, salinity, light, temperature, and other culture conditions (food availability, etc.) were statistically similar between the decreased pH and control treatments. In this study, the pH values showed very little fluctuation, and treatment pH levels were relatively stable over the entire experimental period. These well-controlled pH perturbation conditions allowed us to study the effect of projected nearfuture decreased pH on tube structure and mechanical properties of the tube built by H. elegans. 3.1. Changes in Tube Ultrastructure and Size Parameters under Decreased Ph Condition. The overall impacts of decreased pH conditions on the patterns of ultrastructure are summarized in Supplementary Figure 1 (Supporting Information), while the detailed structural information was shown at a higher magnification in Figure 1. Tube (18-day-old postmetamorphic tubeworms) cultured in the control condition (pH = 8.1) showed three structurally distinguishable layers, namely, spherulitic prismatic structure (SPHP), rounded homogeneous crystal structure (RHC), and irregularly oriented prismatic structure (IOP), in order from the exterior to the lumen (Supplementary Figure 1, Supporting Information). Elemental analysis of a control tube using SEMEDS showed the SPHP layer had a Sr/Ca ratio of 0.119 (molar) and Mg/Ca ratio of 0.050, which was Sr-rich. The middle and inner layers had an observed Sr/Ca ratio of 0.073 and higher Mg/Ca ratio of 0.142 and contained a more prominent Mg peak. The SEM-EDS results suggest the SPHP layer may consist of more aragonite, whereas the RHC and IOP layers nearer to the lumen may contain more calcite. However, in the decreased pH 7.8 treatment, the aragonitic SPHP layer was partially eroded or may not properly form, with a Sr/Ca ratio of 0.079 (molar) and a Mg/Ca ratio of 0.124, and the RHC and IOP contained a greater compositional proportion of C



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Figure 1. Scanning electron micrographs of H. elegans cultured in the (left) control pH 8.1 or (right) pH 7.8 treatment. Cross-sectional view of the tubes accreted in (a) control seawater at pH 8.1 and (b) treatment pH 7.8 were compared for their ultrastructures, as labeled in each representative micrograph; (c and d) magnified images of the treatment effects on the outermost SPHP layers. SPHP, spherulitic prismatic structure; IOP, irregularly oriented prismatic structure; RHC, round homogeneous crystal structure. SEM images of the surface of the shell cross-section etched with 0.5 M EDTA for 1 min.

calcite compared to that of the control group, which had a Sr/ Ca ratio of 0.066 and a Mg/Ca ratio of 0.172. The pH effect altered the ultrastructure and composition of the tube: (1) the three structural layers of the tube became less distinctive from each other (Figure 1b), (2) the aragonitic SPHP layer was partially eroded (Figure 1d), and (3) the overall ultrastructures appeared to contain more open spaces (Figure 1b,d). From the thickness and radius measurements using the SEM images (Supplementary Figure 2b, Supporting Information), the 18-day-old tubes showed a reduction of average thickness by 44.9% (t5 = 3.03, p < 0.05, Supplementary Figure 2b, Supporting Information) and average circular radius by 34.0% (t5 = 3.27, p < 0.05, Supplementary Figure 2b, Supporting Information) when subjected to a decreased pH condition. Tube thickness decreased from 43.58 ± 10.39 μm (pH 8.1 control) to 24.03 ± 4.10 μm (pH 7.8 treatment). Similarly, the radius declined from 217.33 ± 30.22 μm to 143.53 ± 28.56 μm (Supplementary Figure 2b, Supporting Information). 3.2. Tube Mineral Density and Volume Decreased in Response to Decreased pH. The 3D surface features of the tubes in H. elegans were visualized after micro-CT scanning and data reconstruction (Figure 2a−d). The tubes in the control showed richer topographical features including two ridges (white arrows) along the longitudinal tube axis (Figure 2a,c). In contrast, the tubes at pH 7.8 showed fewer observable surface features (Figure 2b,d). Notably, the tubes produced at pH 7.8 had regions detected as “pores”, including the two sides and the point posterior ends of the tube (Figure 2b,d). However, this was caused by the universal setting of detection threshold, suggesting lower-density tubes were produced at pH 7.8. In general, the decreased pH decreased tube volume from 8.16 ± 0.93 × 10−2 mm3 (pH 8.1 control) to 1.72 ± 0.50 × 10−2 mm3 (pH 7.8 treatment) by 78.9% (t5 = 10.75, p < 0.001; Figure 2e). Similarly, tube density reduced from 3.38 ± 0.34 × 10−1 g/mm3 to 1.98 ± 0.27 × 10−1 g/mm3 by 41.4% (t11 = 6.01, p < 0.01; Figure 2e)

Figure 2. Three-dimensional reconstruction of the tubes of H. elegans created from 98−263 microcomputed tomography (CT) images shows the detailed tube structure and topography accreted in (left) control seawater at pH 8.1 and (right) treatment seawater at pH 7.8; (a and b) top view, (c and d) side view. (e) The effects of low pH on the tube mineral (gray bar) volume and (white bar) density as measured from the micro-CT analysis.

3.3. Tubes Constructed under Low pH Had Decreased Homogeneity, Hardness, and Elasticity. Mechanical properties, in terms of hardness and elasticity, were examined using multiple nanoindentation conducted in the middle region of the tube (Supplementary Figure 3a, Supporting Information). These mechanical features distribution over the crosssectional area were summarized in a plot, in terms of normalized depth 0−1 (0 = exterior, 1.0 = lumen; Supplementary Figure 3b, Supporting Information), to compare the impacts of a decreased pH condition (Figure 3). At a decreased pH, overall hardness and elasticity reduced by 80.8% (t3 = 12.75, p < 0.01) and 70.0% (t5 = 13.67, p < 0.001), respectively. The decreased pH treatment reduced hardness from 2.87 ± 0.36 GPa to 0.55 ± 0.03 GPa and elasticity values from 43.02 ± 3.59 GPa to 12.91 ± 1.21 GPa. Furthermore, the distribution pattern of these properties was altered by experimental conditions (Figure 3). In the control, the values of hardness and elasticity distributed more evenly over the normalized depth (Figure 3, ○). In contrast, both values of hardness and elasticity in the tubes produced at pH 7.8 showed a greater variation, with a higher value in the middle region of the normalized depth (Figure 3, ●). In summary, the decreased D



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Figure 3. Over the normalized depth of 0 to 1, (a) hardness and (b) Young’s modulus values were calculated from the load−displacement curves for tubes grown at (○) pH 8.1 and (●) pH 7.8. Figure 4. Effects of pH on the tube breaking force measured by the microforce testing system. (a) A representative load−displacement plot of a tube sample collected from the control pH 8.1 treatments showing the breaking force was ∼9.80 N (black arrow), and (b) the breaking force of the tube produced in control seawater at pH 8.1 and treatment seawater at pH 7.8 were compared (mean ± SD; n = 8).

pH treatment considerably reduced the tube hardness and elasticity values, with more pronounced negative effects on the exterior and lumen sides along the normalized depth of the tube. 3.4. The Tubes Were Easier to Crush at Decreased pH. The crushing force required for tubes cultured in control and treatment conditions were analyzed with the microforce test. The crushing force necessary to bring a tube to the yielding point was measured from a force−displacement curve (Figure 4a). From this measurement, the decreased pH treatment reduced the required crushing force 62.9% (t5 = 8.76, p < 0.001, Figure 4b). The crushing force decreased from 9.05 ± 1.02 N (pH 8.1 control) to only 3.36 ± 0.51 N (pH 7.8 treatment). 3.5. Simulated Tubes at Decreased pH Have Structural Disadvantages. Finite element analysis (FEA) was performed using the measured parameters to understand the mechanical behavior when the tubes face a simulated predatory attack. Representative parameters including the average thickness (control = 43.58 μm; treatment = 24.03 μm), radius (control = 217.33 μm; treatment = 143.53 μm), and the distribution of elasticity, were collected for FEA to study the weakening mechanisms for the control and treatment conditions. FEA models show the distribution of maximum principal stress developed in a representative control and treatment sample under the same loading condition of 3.36 N (Figure 5a,b). The distribution of the stress was similar, where the apical region of the tube had the most concentrated stress on its inner surface (Figure 5a,b), and the maximum principal stress in the control sample (338.8 MPa) was almost half of that in the treatment sample (660.3 MPa). The response of the principal stresses to the simulated applied load suggests different mechanical properties in tube material accreted in the control and treatment conditions (Figure 5c,d). By inputting the crushing force obtained in the microforce testing (control = 9.08 N; treatment = 3.5 N), the

fracture stress of a control tube was higher than in treatment (control = 925 MPa; treatment = 655 MPa; Figure 5c,d). This difference in fracture stress indicates a change in tube material properties due to the treatment effects of decreased pH.

4. DISCUSSION When subjected to the near-future average acidity of seawater pH 7.8, the tubeworms produced intact calcareous tubes of lower density and smaller size in terms of thickness, radius, and tube volume. The tube microstructure and surface topography, when observed under an SEM or microCT, showed notable corrosive action of the decreased pH treatment. The mechanical performance when facing a decreased pH condition was lower in terms of both the multiple nanoindentation tests and crushing tests. For multiple indentation test, the tubes produced at a decreased pH had a weaker innermost and outermost tube layer that may be caused by corrosion, erosion, or dissolution under decreased pH. The crushing force required, the hardness, and the elasticity values were generally lower in tubes produced at pH 7.8. To understand shell mechanical properties in relation to the measured geometry of the tube, we performed finite element analysis (FEA) on the two-dimensional tube models constructed from the average of the measured parameters. The FEA analysis showed a more detailed stress distribution and weakening mechanism of the tube when facing external attack, and show intrinsic differences between the tube materials produced in the control and in the treatment conditions. E



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The impact of a mild reduction in pH seems to have accumulated over a prolonged period. This study demonstrated a greater impact of decreased pH or OA on the 18-day-old tubeworm than the 7-day-old juvenile tubeworms.14 At a decreased pH of 7.8, the tubeworms produced impaired tubes with a less pronounced outer SPHP layer, and less distinct crystalline inner layers that also had a more porous structure (Figure 1b,d). The decreased pH level used in this study is aragonite saturated (Ω > 1), indicating any potential compositional shift would not be due to dissolution but would be mostly due to differences in tube deposition. The tubeworm may strategically favor a compositional shift to a high calcite-toaragonite ratio in the tube when exposed to a decreased pH environment, 14 which could account for the observed alterations in the mechanical properties.56,57 A partial loss of aragonite and/or production of disordered crystal morphologies may result in a more brittle structure dominated by heterogeneously sized calcite.57,58 Aragonite is a more soluble polymorph of CaCO3, and preferential dissolution of aragonite, even in an aragonite-saturated environment, could potentially explain the changes in mechanical properties.59,60 Furthermore, Mg/Ca enrichment could also be used to determine the mechanical strength of the biomineral.61 Several marine invertebrates such as oysters, clams, mussels, and tubeworms are similarly prone to decreased pH and show impaired skeletal morphology and altered mineral composition and ultrastructure.59,60,62,63 For example, aragonite crystal size, shape, orientation, and composition were all significantly altered in corals at pH 7.8.64 Similarly, decreased pH led to impaired ultrastructure in several other marine species.40,65,66 One possibility is that the production of impaired ultrastructure implies a decreasing aragonite saturation state at the site of calcification in response to decreasing environmental pH.67 Mechanical properties of biological materials can be related to crystal size, arrangement, and composition.68 The exterior SPHP layer is predominantly made of aragonitic structures with an ordered arrangement of crystallites. Due to the limitation in precision of EDS, this observation needs to be further verified by other techniques. Nevertheless, Tanur et. al has shown that the adult SPHP layer of a close tubeworm species Hydroides dianthus, is composed of aragonite while the IOP layer is constructed using both Mg-calcite and aragonite.70 This layer was partially eroded in tubes at decreased pH (Figure 1d)14 and tends to have a mixture of both aragonite and calcite crystals at decreased pH (Figure 1d). Studies have shown that changes in ultrastructure can be a direct fortification factor in deciding the overall mechanical properties,69 and this exterior layer is expected to provide high mechanical resilience to external attack and to prevent crack propagation.30,70 This study revealed that this protective SPHP layer faces a challenge at a decreased seawater pH that may lead to a less effective role in mechanical protection. Organic matrix proteins occluded in skeletons regulate crystal size, morphology, and arrangement, which play a significant role in determining the overall mechanical strength.71−74 Therefore, any changes in quantity or quality of crystals or organic matrix proteins under decreased pH may lead to production of weaker tubes. The porous structure in SEM images could also be partially occupied by organic matrix before bleaching, although this possibility needs further verification. Despite the tubes being maintained in ethanol, which is known as a common procedure to maintain organic matrix, future study regarding OA effects on organic matrix

Figure 5. Mechanical effects on H. elegans tube structure. Twodimensional finite element models based on elasticity values obtained from multiple indentation experiment (n = 8). Both the model for (top) control and (bottom) treatment samples are subjected to the same loads and boundary conditions mimicking biological attacks (black arrows). The von Mises stresses are displayed at the same scale for comparative purposes (warm colors represent high stress, and cool colors represent low stress). The two models have different stress patterns. (a) Model based on tube produced at pH 8.1 showed a more dispersed stress throughout the tube structure. (b) Model based on tube produced at pH 7.8 showed greater regional stress stratification. (c) Stress development versus forced applied curves on potential zones of weakness are compared at () top, (− −) side, and (---) bottom tube attachment region. F



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Although ecology and larval biology of this worm has been intensively studied, only few studies attempted predation on them by fouling community associated flatworms, fishes, crustacean, and sea urchins.55 Nevertheless, tubes of this worm are likely to be broken by flatworms, sea urchins, crabs, fishes, and various molluscs. To withstand a predatory pressure from aggressive biters such as fishes and crabs, the tubeworms are expected to have mechanically strong-enough tubes. Detailed mechanical measurements of tube ultrastructure and overall mechanical performance would be a useful diagnostic method to enable the prediction of the impact of humaninduced ocean acidification effects on marine organisms and to advance our understanding of environment−biomineral interaction. The weakening of the protective tube is likely to increase the vulnerability to predation and to reduce chances of survival in a stressful environment with negative consequences to coastal community in the immediate near future.31

formation and quantity will be appreciated to reflect more closely to real mechanical properties of living polychaetes and will enhance the understanding of the in situ organic−mineral interaction under OA. Observed negative effects of decreased pH on tube mechanical strength is apparently not unique to tubewormssimilar negative effects have also been observed in corals,75 clams,76 oysters, and mussels.17,77,78 Size parameters such as tube thickness, radius, and volume were shown to decrease under pH 7.8. Furthermore, the observed reduction in density suggests the calcified materials may be inherently less resistant. Field observation strongly suggests that marine organisms tend to produce lighter (less dense) calcareous structures in decreased pH environments.79 Conventional size measurements overlooked any potential decline in mineral density, which may lead to overestimation of mechanical properties. The more detailed investigation of multiple nanoindentation showed that the exterior and innermost layers of the tube had poor mechanical properties compared to the middle layer when built at pH 7.8 (Figure 3). This result suggests that the outermost and innermost surfaces of the tube may be prone to severe damage under decreased pH. Most calcifying marine organisms have the ability to secrete a protective organic layer to protect the calcified structure from environmental stressors such as decreased pH.59 Tubeworms are able to produce a similar protective organic layer, but it appears to be considerably thinner than in many other organisms.59,70 This organic layer only occurs in lumen layer, leaving the minerals on the external tube surface in contact with the seawater.80 Organisms living near the naturally acidified ecosystems of CO2 vents are reported to have eroded shells as a consequence of limited protection by the organic layer.81 Some serpulids including Hydroides secrete a thin organic lining within the mineral structures of the tube,82 which may retard the process of corrosion in the acidified water to a certain extent. In addition, for even above aragonite saturation levels, decreased environmental pH may ultimately reduce intracellular pH if the animal does not have adequate homeostatic mechanisms.83 Under this situation, the innermost layer of the shell would also be exposed to corrosive pH levels due to accumulated metabolic acid.84 Therefore, both the external and internal surfaces of the tube can be drastically affected by decreased pH and lead to the reduced mechanical strength, as we observed in our results. Crystal microstructures are the building blocks in the calcified structures,68 which would be energetically expensive to maintain and prone to be altered in decreased pH environment.83 Besides, The calcifying serpulid tubeworms are a diverse family that differs in both polymorphs and ultrastructure.50 However, the FEA approach allows a generalizable method to understand the mechanical response of diverse calcifying marine organisms to external predator attack. Size parameters with elasticity measurements are important for understanding the pattern of stress development and estimating changes in the material properties in the computational FEA method.52,85,86 As a result, the structural function of each component in structure can be evaluated by analyzing the stress distribution. Moreover, induced changes in shell shapes can be further interpreted to depict the stress distribution and fracture mechanism in the calcareous structure as a result of the treatment effects. The findings of our study strongly suggest that tubeworms will be vulnerable to projected near-future ocean acidification.

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank Y. Y. Chui (University of Hong Kong, HKU) for sectioning and HKU-EMU for the SEM analysis and Dr. Kelvin Yeung and Tony Liu for their help on microCT work. We thank our laboratory ocean acidification advisors, Richard Bellerby, Maria Byrne, Mary Sewell, Jason Hall-Spencer, Sam Dupont, Ishimatsu Atsushi, and other members of the ISOACC group for their valuable discussions during the course of this project. We also thank Maggie Cusack (University of Glasgow) and Andrew Mount (University of Clemson) for their valuable contributions during our tube analysis. This study was funded by three GRF grants from the HKSAR-RGC (Grant Numbers: 705511P, 705112P, and 17304914).

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