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Shell Hardness and Compressive Strength of the Eastern Oyster, Crassostrea virginica, and the Asian Oyster, Crassostrea ariakensis.

Introduction

Adult oysters possess two calcareous valves connected by a hinge ligament that acts as a spring and allows an oyster to separate the valves (gape), thereby permitting an exchange between the internal and external environments (Carriker, 1996). Each valve is composed of three primary layers. The outermost layer is the periostracum, which primarily consists of an organic (conchiolin) matrix and protects the shell from corrosion (Galtsoff, 1954, 1964; Bottjer and Carter, 1980; Carriker, 1996; Fig. 1). The middle layer is the prismatic layer, which contains curved, wedge-shaped prisms of calcite crystals within a conchiolin matrix (Galt-soft 1954, 1964; Mount et al., 2004). The innermost layer, nearest the pallial space, is the calcite-ostracom layer. also known as the foliated layer. The foliated layer constitutes the majority of the shell and comprises sheets of calcite that are associated with providing shell strength (Galtsoff, 1954, 1964; Chateigner et al., 2002; Mount et al., 2004). Since the foliated layer is composed of parallel sheets of calcite crystals, its microscopic structure is similar to that of the nacre layer of aragonite structures (Checa et czl., 2007). Within oyster shells, the foliated layer acts as the main defense that predators must penetrate to access oyster tissue. Overall, the shells assist in survival, as they provide space for internal organs and shield tissues from outside forces that include predators as well as environmental stressors such as salinity changes, desiccation, and anoxic or contaminated water (Carriker, 1996; White and Wilson, 1996). Therefore, understanding the properties of the shells, in particular the foliated layer, can contribute to understanding oyster predation defenses and survival. Ultimately, the mechanical defense provided by the shells enables oysters to perform essential ecosystem services such as habitat formation, water filtration, and benthic-pelagic coupling in marine and estuarine systems (see Newell, 1988; Meyer and Townsend, 2000; Steimle and Zetlin, 2000; Porter et at., 2004; Grabowski et al., 2005; see Gerakii et at., 2009 for a review of oyster ecosystem services).

Throughout the past century the Chesapeake Bay population of the Eastern oyster, Crassostrea virginica (Gmelin, 1791), has been in decline due to over-harvesting, habitat destruction, and the introduction and spread of parasitic diseases such as Dermo, caused by Perkinsus marinus (Mackin, Owen, and Collier) Levine, 1978 (Newell, 1988; Rothschild et al., 1994; Ford and Tripp, 1996). However, the morphologically and taxonomically similar Asian oyster, Crassostrea ariakensis (Fujita, 1913), is more resistant to P. marinus infection and more resilient to its effects when infected (Calvo et al., 2001; NRC, 2004). This led to the consideration of C. ariakensis for introduction to the Chesapeake Bay to augment the native population, thereby aiding in ecosystem services and providing an alternative species for the commercial oyster industry. The potential introduction of C. ariakensis to part of the native range of C. virginica resulted in numerous studies comparing the anatomy and physiology of these two species (e.g., Calvo et al., 2001; Matsche and Barker, 2006; Harding, 2007; Harlan, 2007; Lombardi. 2012; Lombardi et of., 2013). During one such study, it was observed that when notching shells with a grinding wheel, considerable more effort was required for C. virginica than for C. ariakensis (Lombardi and Paynter, unpubl.). This observation illustrated the need for a more in-depth investigation of the mechanical properties of the valves of these oysters, in order to understand the mechanisms for these apparent differences in strength and durability that may translate to differences in survival.

In this study, we investigate factors potentially affecting oyster shell strength, including age, species, and shell wetness, and we discuss the implications of these factors for predation pressures. The mechanical properties of hardness (resistance to irreversible deformation) and compressive strength (force necessary to produce a crack in a material) of oyster shells were measured in various age classes of C'. ariakensis and C. virginica. Since oyster shell layers accumulate as the animal grows, different layers may have different properties. Therefore, shells were also tested for differences in the hardness between superficial and deep foliated layers. Further, the compressive load at fracture, a metric of the ability to withstand pressure without cracking, was measured on the whole shell.

On the basis of perceived shell durability, we hypothesized that C. virginica shells would be more resistant to fracture and indentation, and thus harder and able to withstand a higher compressive force than C. ariakensis shells. We further hypothesized that age would be correlated with thickness and that older shells would be able to withstand a higher compressive force without cracking than younger oyster shells. The results of this study provide insight into the shell properties of two similar oyster species and may contribute to an understanding of mechanisms for defense against predators and for survival.

Materials and Methods

Oyster preparation

Crassostrea virginica individuals (ages 1, 4, 6, and 9 years) were obtained from hatchery-planted bars in the Choptank River in the northern Chesapeake Bay, and Cras-sostrea ariakensis individuals (ages 4 and 6 years) were obtained from University of Maryland Center for Environmental Science's Horn Point Laboratory Oyster Hatchery program. Each oyster was scrubbed with a wire brush to remove any debris and epifauna and then shucked to isolate shells from oyster tissue. Shell height (mm) and total mass (g) of each individual were also measured.

Hardness

The hardness, measured in gigapascals (GPa), of four C. virginica shells from each age class (1, 4, 6, and 9 years) and four C. ariakensis shells from each age class (4 and 6 years) was tested. A diamond wheel was used to cut two pieces (about 10 mm by 20 mm) from the flattest part of each valve. One piece was used to test hardness at the deepest foliated layer, and the second piece was used to test hardness at the most superficial foliated layer. In all samples, the area where the adductor muscle attached to the valve was not used so as to maintain consistent valve sampling.

Each valve sample was embedded in resin and polished with a Buehler EcoMet 4000 Variable Speed Grinder-Polisher (Lake Bluff, IL) with water or suspensions of abrasive particles between the samples and grinding pads, in order to facilitate the collection of clear microindentations (Chinn, 2002). Each sample was polished for 10-40 min at each grit size (400, 600, and 1200 grit with water, microcloth with Buehler TexMet 1.0-[micro]m suspension, and microcloth with 0.5-[micro]m diamond suspension). Between each grit size, all samples were placed in a sonicator for 5 min and then observed under a microscope to inspect for uniform surface scratches to determine if the sample was ready to progress to the next grit size. After the finest grit polishing, the samples were mirror-smooth (Fig. 2A).

Each valve sample was taken to the National Institute of Standards and Technology where five indents on each sample were made using a Vickers pyramidal diamond micro-indenter (Zwick, GA; Fig. 2B). Each indent image (Fig. 2C) was captured using a Leco Olympus PMG3 microscope (Olympus, PA). Hardness values were calculated using the standard Vickers hardness (HV) equation:

HV = 1.854 x [Fld.sup.2],

where F is the applied force (1 kg and 9.8 m/[s.sup.2]) and d is the mean length (mm) of the indentation diagonals. Mean hardness values for each sample were calculated.

Analysis of hardness between superficial and deep foliated layers, species, and age (4- and 6-year-olds) was performed using a fixed-factor split-plot analysis of variance (ANOVA), with oyster age as the main plot and each foliated layer as the subplot. A separate one-way fixed-factor ANOVA was conducted to test for intraspecific hardness differences among dry specimens of C. virginica at different ages (1, 4, 6, and 9 years) using the foliated layer with the lowest hardness value.

Compressive strength

Intraspecific and interspecific comparisons. Interspecific analyses based on age classes (4 and 6 years) were conducted using 10 dry shells of each species. Additionally. 10 dry C. virginica shells aged one and nine were used for intraspecific analyses. Before the compressive strength was measured, shell thickness within 5 mm of the ventral shell margin of the curved valve was measured using digital calipers (ram). The compressive force, measured in newtons (N), needed to fracture each shell was determined by using an Instron load compression machine with a flat load cell (Instron, PA). The left and right valves of each shell were realigned to mimic a closed live oyster. The flat right valve was placed on the base of the machine, while the cupped left valve pointed upward with the tallest point of the shell aligned with the center of the machine. Compressive strength was assessed by increasing the force until the shell cracked and there was a major drop in load compression force. After the drop in force was observed, the oyster was examined to confirm the presence of a crack. In all samples, the crack was visually identified and went through all layers of the shell. Typically both valves were cracked, though on occasion only the upper, cupped valve cracked. We observed complete radial cracks that originated in the center of the shell and spread outward to the edge.

A two-way fixed-factor ANOVA was used to test for differences in compressive strength based on age and species and to test for any interactions between the two. Additionally, a one-way fixed-factor ANOVA was used to test for differences in compressive strength between all ages (1, 4, 6, 9 years) of C. virginica. To determine if thickness could explain the patterns observed, we tested whether thickness differed between age classes or species by using a two-way fixed-factor ANOVA and two separate analysis of covariance (ANCOVA) models (one with species as a factor and a second with age class) with thickness as a covariate.

Wet versus dry shells. The relationship between shell wetness and compressive strength was also investigated using six-year-old oyster shells; five shells of each species were kept wet, and five shells of each species were placed in a 60 [degrees]C oven for 72 h to dry. Dry shells were stored in a desiccator at room temperature (about 24 [degrees]V) until compressive strength data were collected using the procedure described in the previous section on Intraspecific and inter-specific comparisons. A separate two-way fixed-factor ANOVA was used to test for significant differences in compressive strength between wet and dry shells, species, and any interaction between the two factors.

Shell density

Density was measured in 10 C. virginica shells for each age (1, 4, 6, and 9 years) and 10 C. ariakensis shells for each age (4 and 6 years). Shells measured for density were different shells than those used for either compressive strength or hardness tests but were from the same source and were conditioned identically. The volume of each pair of valves was calculated by displacement using graduated cylinders, and the dry mass of the shells weighed on a microbalance. Using these values, density (g/m1) was calculated for each shell. A separate two-way fixed-factor ANOVA with age and species as factors was used to compare shell densities.

For all comparisons, statistical analyses were performed in JMP (JMF', NC) and R 2.11.1 (R Core Team, 2013). In some cases, log-transformed data were used to meet normality or homogeneity of variance assumptions. Since the P values between transformed and non-transformed data were comparable and the accept/reject decisions were the same, results are presented on non-transformed data to ease interpretation.

Results

Hardness

Intraspecific Crassostrea virginica comparisons (ages 1, 4, 6, 9 years). The mean ([+ or -]SEM) shell hardness values for one-year-old individuals of C. virginica were 1.17 [+ or -] 0.17 GPa, 1.55 -[+ or -] 0.22 GPa for four-year-olds, 1.47 -[+ or -] 0.16 GPa for six-year-olds, and 1.20 [+ or -] 0.14 GPa for nine-year-olds. Hardness values were not significantly different between age classes of C. virginica (P = 0.368). As there was no significant difference in hardness between outer and inner foliated layers (P = 0.274, see the following Results section on interspecific comparisons), the intraspecific analysis was performed using the lower hardness value from each shell. This was done because most predators would need to penetrate only one valve, and therefore we chose to use the weaker valve.

Interspecific comparison (ages 4 and 6 years). When we performed the split-plot analysis, no significant two-way or three-way interactions were found for shell hardness between age, foliated layer, or species (in all comparisons P > 0.05). Crassostrea ariakensis exhibited a shell hardness of 1.45 [+ or -] 0.13 GPa, which was not significantly different from that of C. virginica shells (1.77 [+ or -] 0.13 GPa; P = 0.090). Shell hardness values did not significantly vary between four-year-old (1.64 [+ or -] 0.15 GPa) and six-year-old oysters (1.57 [+ or -] 0.12 GPa; P = 0.635) or between foliated layers ([[mu].sub.deep]= 1.50 [+ or -] 0.12 GPa; [[mu].sub.superticial] = 1.72 [+ or -] 0.14 GPa; P = 0.274). It should be noted that C. virginica shell values differ from those in the intraspecific analysis (previous Results section), because the intraspecific analysis used only one foliated layer per oyster, whereas this analysis used two samples per shell.

Compressive strength

Intraspecific Crassostrea virginica comparisons. No significant difference in compressive strength was found between one- (853.9 [+ or -] 169.1 N) and four-year-old C. virginica shells (1089.8 [+ or -] 90.8 N; P = 0.235). However, a significant increase in compressive force was needed to crack both six-and nine-year-old shells (mean forces were 3896.1 [+ or -] 560.7 N and 3513.1 [+ or -] 476.3 N, respectively) compared to one- and four-year-old shells (mean forces were 853.9 [+ or -] 169.1 N and 1089.8 [+ or -] 90.8 N; P < 0.001, P < 0.001, respectively).

Interspecific analysis (ages 4 and 6 years)

A significant interaction was found between age and species for compressive strength (P = 0.001; Fig. 3A). The ability to withstand compressive force did not statistically vary between four- (971.4 [+ or -] 84.1 N) and six- (1522.0 [+ or -] 315.1 N) year-old C. ariakensis (P = 0.109). However, six-year-old C. virginica shells were able to withstand a significantly higher compressive force (3896.1 [+ or -] 560.7 N) than both four- (P < 0.001) and six- (P = 0.002) year-old C. ariakensis shells. Compressive strength did not differ between shells of four-year-old C. virginica or age four- or six-year-old C. ariakensis (P = 0.538).

Thickness, density, and wetness. A significant exponential relationship between the compressive force required to produce a crack (y) and shell thickness (x) was found (y = [523.6e.sup.0.381x], [R.sup.2] = 0.4657; P < 0.001; Fig. 4)--thicker oysters withstood higher compressive force. ANOVA results indicated that thickness varied on the basis of species and age class (significant interaction between species and age, P = 0.003). At age four, the thickness of C. virginica shells (2.47 [+ or -] 0.26 mm) was not significantly different from that of C. ariakensis (2.07 [+ or -] 0.24 mm; P = 0.275); however, at age six, C. virginica shells were more than twice as thick as C. ariakensis shells ([[mu].sub.c. ariakensis] = 2.01 [+ or -] 0.38 mm; virginica = 4.53 [+ or -] 0.42 mm; P < 0.001). ANCOVA indicated no significant interaction between thickness and age (P = 0.172) or thickness and species (P = 0.871) and that in each case thickness had a significant effect (P = 0.001; P = 0.002, respectively). After accounting for thickness using ANCOVA, species were no longer different (P = 0.358), but a significant trend persisted with age--valves of older oysters had a higher compressive strength than younger valves (P = 0.004).

When analyzing density, no significant interaction between species and age was found (P = 0.923). Age also had no effect on density (P = 0.876), but C. virginica shells exhibited a mean density of 2.18 [+ or -] 0.06 g/ml, which was more dense than C. ariakensis shells (1.44 [+ or -] 0.12 g/mL; Fig. 3C; P < 0.001). No interaction was found between wetness and species (P = 0.930), and the force needed to produce a crack did not differ between wet (2512.8 [+ or -] 441.8 N) and dry (2191.5 [+ or -] 532.7 N) shells (P = 0.547).

Discussion

This study investigated the mechanical properties of compressive strength and hardness across different ages of adult Crassostrea virginica and C. ariakensis shells. We found no differences in shell hardness by age, layer (innermost or outermost foliated layer), or species. In view of the hardness data, we believe the foliated layers of adult C. virginica and C. ariakensis likely have similar compositions, giving rise to similar strength and defenses. As similar calcite configuration likely yields similar strength, our findings are in agreement with those of Checa et at. (2007), who found that the crystallographic structure of the foliated layer had a consistent arrangement across bivalve species. Further, compressive force data revealed that the compression strength of C. virginica shells increased with age, but no difference was found in compression strength between C. ariakensis age classes. Interspecifically, ages six and nine of C. virginica could withstand significantly more compression force than either age class (4 or 6 years) of C. ariak-ensis.

In particular, the compression strength of six-year-old C. ariakensis shells was 61% lower than that of six-year-old C. virginica shells; this difference is similar to the one (64%) observed between spat (oysters less than a year old) of these species (Newell et at., 2007). Interestingly, however, at age four no significant difference (-10%) existed between species. Therefore, the ratio of compressive strength between C. virginica and C. ariakensis individuals likely varies by age. While the reason for this is unknown, it may reflect differences in growth pattern, growth rate, energy allocation, or life-history strategies between these species.

Compressive strength was not different between species at age four years, but six-year-old C. virginica shells withstood a higher compressive force than six-year-old C. ari-akensis, a pattern that may be explained by differences in density and thickness. The shells of C. ariakensis were 34% less dense than those of C. virginica. Regression analysis indicated a significant relationship between valve thickness and compressive strength, and six-year-old C. virginica had the thickest valves. Additionally, when an ANCOVA was performed with thickness as a covariate, species no longer had an effect on compression strength. Local curvatures notwithstanding, the dependance of loads required for fractures originating in the interior section of the shells and other material are dependent on the square of thickness, as seen in work presented in Qasim et al. (2006, 2007). Therefore, the protective nature of shell thickness versus the load required to fracture the shell under a point load reveals that small attenuations in shell thickness impact fracture loads by a squared relation. However, our data do not fully adhere to this pattern. We believe the discrepancy is due to the shape of the shells, as curvature can distribute the load over the entire structure, thereby influencing the compressive strength (Sayegh and Dong, 1970). Therefore, the mechanical defenses of shells likely include the foliated layer structure and the curve and thickness of the shell. In summary. we believe that the compressive strength differences between species can be primarily explained by differences in shell thicknesses and density; however, those factors do not explain ontogenetic differences based on age.

Since many predators penetrate through the shell to access internal oyster tissue (White and Wilson, 1996), identifying differences in shell strength by age and species could have implications for restoration and management of the native population and the stability of the fishing industry. Therefore, the differences in strength, illustrated by the ability to withstand compressive force, between C. ariak-ensis and C. virginica could translate into differences in predation. Although predators vary by location, common predators of oysters include fish (namely drums, sheeps-heads, rays, and skates); crabs; snails (especially drills); sea stars; and flatworms (Mackin, 1959). However, species have different mechanical abilities to penetrate the shell. For instance, the cownose ray, Rhinoptera bonasus (Mitchill, 1815), is capable of bite forces of about 220 N (Maschner, 2000); and the bite of the large black drum, Pogonias cromis (Linnaeus, 1766), can reach up to 1250 N (Grubich, 2005). Further, the rock crab, Cancer irroratus Say, 1817, has demonstrated chelae crushing forces of up to 75 N (Block and Rebach, 1998); and the blue crab, Callinectes sapidus Rathbun, 1896, has exhibited a mean chelae force of 27 N (Singh et aL, 2000). In contrast, the mean compressive strength of C. virginica spat ranged from 51 to 71 N and that of C. ariakensis from 18 to 25 N, depending on the presence of predators (Newell et al., 2007); in this study we found the compressive force needed to crack the shells of one- and four-year-old C. virginica and four-year-old C. ariakensis was about 1000 N, whereas six- and nine-year-old C. virginica withstood more than 3500 N. Thus, if a predator attacks the center of the shell, then both spat and adult C. virginica and C. ariakensis should be able to withstand cownose ray, rock crab, and blue crab predation, but C. ariakensis and young (ages four and younger) C. virginica are still vulnerable to drum predation.

Another factor that may influence shell properties is the ratio of chalky deposits to foliated sheets within the valves. Naturally interspersed between foliated sheets are chalky deposits (Galtsoff, 1964; Carriker, 1996). Although both chalky and non-chalky layers are made of similar chemical elements, they have different chemical compositions, and chalky material is more porous than the folia (Yoon et at., 2009; Lee et al., 2011). Galtsoff (1964) found that 50% of C'. virginica samples had no chalky patches, and only 3% had more than 75% of the shell composed of such deposits. However, no studies have quantified the chalky proportions in C. ariakensis. In this study we were interested in assessing the hardness of the foliated layer; however, while collecting data we encountered regions of high variability within C. ariakensis. We believe these represented samples taken in the chalky layer, and we excluded them from analysis (Appendix Fig. 1). Although some discrepancy exists in the literature (Lee et aL, 2011), Yoon et at. (2009) and Ullmann et at. (2010) found the chalky layer of Cras-sostrea oysters species to be less dense than the foliated layers. Therefore, since regions of high variability were found only in C. ariakensis valves that were also less dense than those of C. virginica, we hypothesize that C. ariakensis may have a higher proportion of chalky material than C. virginica. A difference in this proportion could also explain the differences in strength and durability observed at the macroscopic level--that is, the greater difficulty that Lombardi and Paynter (unpubl. data) experienced in grinding C'. virginica shells compared to C ariakensis shells. Despite providing less strength, a higher proportion of chalky material may be advantageous in an area dominated by gape-or claw-limited predators because chalk deposits may increase shell thickness at minimal cost to the oyster. Thus, different proportions of chalky material to folia likely influence the mechanical defenses the shell provides and may indicate different predation styles and pressures in native ranges. Overall, the shells of C. ariakensis may be less durable and strong due to a greater proportion of chalky material, though further studies are needed to test this hypothesis.

In summary, we found that the shells of six- and nine-year-old specimens of C. virginica withstood significantly higher compression force than any age class of C. ariakensis studied (4- and 6-year-old). The higher compressive strength observed for C. virginica shells at age six is likely due to thicker valves and denser shells compared to C. ariakensis. We found no differences in hardness values between any age class, foliated layer, or species; however, differences in density and observed variations in hardness within C. ariakensis (Appendix Fig. 1) may indicate different shell compositions between species. The findings of this research can be applied to oyster management decisions, as well as to oyster-inspired bioceramic technology. Moreover, this work contributes to an understanding of shell mechanics and sheds light on similarities and differences between two closely related species while identifying potential differences in predation vulnerability.

Acknowledgments

Support for this research was provided by grants to the University of Maryland from the Oyster Recovery Partnership and the Howard Hughes Medical Institute Undergraduate Science Education Program. This project would not have been possible without the encouragement, assistance, and expertise of Peter Lucas, Erin Vogel, and Shannon McFarlin from George Washington University. We thank Brian Lawn from the National Institute of Standards and Technology for his materials science expertise and guidance. Further, we are grateful to Adriane Michaelis, Karen Kesler, Rebecca Kulp, Emma Weaver, and Drew Needham for their assistance with shell polishing and data collection, and to Robert Bonenberger, Vincent Politano, Jeffrey Jensen, and William Higgins for their academic and logistical help. Additionally, we thank the Horn Point Oyster Hatchery program for supplying the Crassostrea ariakensis used in this study. We also thank the editor and two anonymous reviewers for constructive comments that helped strengthen the manuscript.

Reference: Biol. Bull. 225: 175-183. (December 2013) [c] 2013 Marine Biological Laboratory

Received 29 March 2013; accepted 26 November 2013.

* To whom correspondence should be addressed. E-mail: SaraAnn Lombardi@grnail.com

[dagger] Current address: Department of Environmental Science, American University, 4400 Massachusetts Ave. NW, Washington, DC 20016.

[paragraph] Current address: Department of Natural Sciences, Hawaii Pacific University, 45-045 Kamehameha Highway, Kaneohe, HI 96744.

[section] Current address: Department of Biological Sciences, Marshall University, 1 John Marshall Drive, Science Building Room 350, Huntington, WV 25755.

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Appendix

Differences in density and observed variations in hardness within Crassostrea ariakensis

SARA A. LOMBARDI (1), * ([dagger]), GRACE D. CHON (1), ([paragraph]), JAMES JIN-WU LEE (2), ([section]), HILLARY A. LANE (1), AND KENNEDY T. PAYNTER (1), (3)

(1) Department of Biology, University of Maryland, College Park, College Park, Maryland 20742; (2) Ceramics Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899; and (3) Chesapeake Biological Laboratory, University of Maryland Center for Environmental Science, P.O. Box 38, 1 Williams Street, Solomons, Maryland 20688
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Author:Lombardi, Sara A.; Chon, Grace D.; Lee, James Jin-Wu; Lane, Hillary A.; Paynter, Kennedy T.
Publication:The Biological Bulletin
Article Type:Report
Geographic Code:1USA
Date:Dec 1, 2013
Words:5464
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