Three-Dimensional Structure of the Shell Plate Assembly of the Chiton Tonicella Marmorea and Its Biomechanical Consequences

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Journal of Structural Biology 177 (2012) 314–328

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Journal of Structural Biology

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Three-dimensional structure of the shell plate assembly of the chiton Tonicella marmorea and its biomechanical consequences

Matthew J. Connors a, Hermann Ehrlich b, Martin Hog b, Clemence Godeffroy b, Sergio Araya c, Ilan Kallai d, ⇑ Dan Gazit d,e, Mary Boyce f, Christine Ortiz a, a Department of Materials Science and Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA b Institute of Bioanalytical Chemistry, Dresden University of Technology, 01069 Dresden, Germany c Department of Architecture, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA d Skeletal Biotech Laboratory, The Hebrew University – Hadassah Faculty of Dental Medicine, Ein Kerem, Jerusalem 91120, Israel e Department of Surgery and Cedars-Sinai Regenerative Medicine Institute (CS-RMI), Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA f Department of Mechanical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA article info abstract

Article history: This study investigates the three-dimensional structure of the eight plate exoskeletal (shell) assembly of Received 2 September 2011 the chiton Tonicella marmorea. X-ray micro-computed tomography and 3D printing elucidate the mech- Received in revised form 12 December 2011 anism of conformational change from a passive (slightly curved, attached to surface) to a defensive Accepted 14 December 2011 (rolled, detached from surface) state of the plate assembly. The passive and defensive conformations Available online 10 January 2012 exhibited differences in longitudinal curvature index (0.43 vs. 0.70), average plate-to-plate overlap (62% vs. 48%), cross-sectional overlap heterogeneity (60–82.5% vs. 0–90%, fourth plate), and plate- Keywords: to-plate separation distance (100% increase in normalized separation distance between plates 4 and 5), Chiton respectively. The plate-to-plate interconnections consist of two rigid plates joined by a compliant, actu- Shell microstructure Exoskeleton ating muscle, analogous to a geometrically structured shear lap joint. This work provides an understand- Natural armor ing of how T. marmorea achieves the balance between mobility and protection. In the passive state, the Micro-computed tomography morphometry of the plates and plate-to-plate interconnections results in an approximately continuous curvature and constant armor thickness, resulting in limited mobility but maximum protection. In the defensive state, the underlying soft tissues gain protection and the chiton gains mobility through tidal ﬂow, but regions of vulnerability open dorsally, due to the increase in plate-to-plate separation and decrease in plate-to-plate overlap. Lastly, experiments using optical and scanning electron microscopy, mercury porosimetry, and Fourier-transform infrared spectroscopy explore the microstructure and spatial distribution of the six layers within the intermediate plates, the role of multilayering in resisting predatory attacks, and the detection of chitin as a major component of the intra-plate organic matrix and girdle. Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction symmetric, and generally range from a few mm to 15 cm in length (Eernisse and Reynolds, 1994). Chitons are of great interest from a Chitons (Polyplacophorans) are marine mollusks found biomechanical perspective because instead of a single continuous worldwide in all seas with more than 940 extant (Schwabe, shell, they possess an assembly of eight dorsal aragonite-based 2005; Stebbins and Eernisse, 2009) and 430 fossil species (Puchal- plates (valves) (Fig. 1a). The first (head) and eighth (tail) plates ski et al., 2008) which extend to the late Cambrian (Runnegar et al., are semicircular in outline while the intermediate plates are ‘‘but- 1979). Chitons are one of the earliest diverging groups of living terfly’’-shaped. These plates provide protection while still allowing mollusks and are often referred to as ‘‘living fossils’’ since for some degree of flexibility during locomotion over uneven, their body plan has not significantly changed for over 300 million rough surfaces as well as when rolling defensively into a ball-like years (Sigwart, 2009). They are typically oval in shape, bilaterally conformation (Fig. 1b) if dislodged from a surface. In many species, the head plate nearly touches the tail plate in the rolled state (Vermeij, 1993). This defensive conformation covers and protects ⇑ Corresponding author. Department of Materials Science and Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Room 13-4022, the ventral side of the chiton from imminent predation, and may Cambridge, MA 02139, USA. Fax: +1 617 253 5620. allow it to be carried out of harm’s way by a passing wave to a E-mail address: [email protected] (C. Ortiz). more favorable location to right itself (Arey and Crozier, 1919;

2) Dorsal Mesostracum: a thin simple crossed layer which may (a) 5 4 3 be divideddorsoventrallyinto two sub-layers by a row of aes- 6 2 thete channels. 3) Articulamentum: a composite prismatic layer that constitutes 7 the bulk of the apophyses and insertion plates. 4) Ventral Mesostracum: a simple crossed-lamellar layer. The dorsal and ventral mesostracum layers are collectively 8 1 referred to the as the Pallial Myostracum. 5) Anterior Myostracum: a granular or irregular simple prismatic layer which stems from the impressions of the oblique Girdle and post-apophysial latero-pedal muscles. Posterior Anterior 6) Posterior Myostracum: a granular or irregular simple pris- 0.5 cm x matic layer which lies in the impression of the transverse • z muscle. 7) Hypostracum: a simple crossed-lamellar layer that constitutes the bulk of the central callus. y

The objectives of this study were to (1) to quantify the three- (b) dimensional geometric structure of individual plates, as well as of the entire plate assemblies of chitons in passive (slightly curved, attached to surface) and defensive (rolled, detached from surface) conformations, (2) to determine the spatial distribution of microstructures present in intermediate plates and microstructural orientations relative to macroscopic plate dimensions, (3) to elucidate relationships between the internal multilayering and microstructures of the internal plates (‘‘inherent materiality’’) and the larger length scale geometric design to better understand Girdle the balance between threat protection and biomechanical mobility, and (4) to gain insights into the functional and physiological consequences related to survivability of the segmented exoskeleton (structurally, not biologically). To accomplish these goals, the Fig.1. Images of Tonicella marmorea; (a) side view optical microscopy image of a chiton Tonicella marmorea was analyzed by X-ray micro-computed dried shell; (b) side view photograph of a recently thawed chiton in a defensive, tomography (microCT) to quantify biomechanically-relevant fea- curved posture. tures such as the three-dimensional geometry of the individual plates, the inter-plate registration and overlap, and the curvature and continuity of the entire plate assemblies. Three-dimensional Eernisse, 2007). The curvature of the plate assembly is most often printing was employed to create a scaled-up macroscopic synthetic convex, although less frequent concave curvatures have been ob- prototype directly from the microCT data to elucidate the detailed served. Convex flexure is provided by the ‘‘enrolling’’ muscle, plate morphometry and to better visualize the inter-plate articula- which encircles the shell underneath the outer margin of the plates tion. The intra-plate microstructure was characterized via mercury (Haszprunar and Wanninger, 2000). Plates 2–8 possess two ante- porosimetry, optical microscopy, scanning electron microscopy rior projections, the apophyses (sutural laminae), which overlap (SEM), and X-ray diffraction (XRD). Fourier-transform infrared with the ventral surface of the adjacent anterior plate. Transverse spectroscopy (FTIR) and amino acid analysis (AAA) were used to muscles lie in the overlapping region between neighboring plates identify and compare the composition of organic materials from and connect them together (Wingstrand, 1985). Also extending the girdle, shell plates, and inter-plate region. The obtained results between the shell plates is a thin cuticular layer (Beedham and are discussed in the context of other species with articulating nat- Trueman, 1967), which laterally attaches to a thick chitinous cuti- ural armor, in particular the balance between the local protection cle that covers a leathery girdle structure surrounding the plates. mechanisms of the individual plates and the larger length scale de- Resistance to penetration is provided by the internal, multilay- sign principles of the plate-to-plate interconnections. The new sci- ered microstructure of the individual exoskeletal units (Bruet et al., entific information obtained holds potential for comparative 2008), in this case the chiton plates, which are known to possess morphometric analyses of chitons, as well as for the development one of the most complex multilayered structures of any mollusk of improved bio-inspired protective materials, for example with re- (Bøggild, 1930). The multilayered structure of the plates have been gards to human extremity protection (Arciszewski and Cornell, studied by a number of authors including Haas (1972, 1976), Laghi 2006; Bruet et al., 2008; Ortiz and Boyce, 2008; Song et al., 2010; and Russo (1978, 1980), Baxter and Jones (1981), Kreusch (1981), Yao et al., 2010). Poulicek and Kreusch (1983), Carter and Hall (1990), Currie (1992), and Chen (2010). In addition to the periostracum, an outer 2. Materials and methods thin organic layer that is often eroded away on older shells, chiton shell plates are generally composed of seven aragonite-based lay- 2.1. Samples and terminology ers as follows (described from outer to inner): Chitons were collected on the coast of Pembroke, Maine and 1) Tegmentum: a granular or irregular simple prismatic layer stored frozen until experimentation. When needed, individual which is penetrated by the aesthetes, an intricate network plates were separated using forceps and razor blades. Plates were of tissue-filled channels that open on each plates’ dorsal sur- cleaned by sonication (Branson 1510, Danbury, CT) in deionized face as sensory organs (Vendrasco et al., 2008). water twice for approximately 45 s. 316 M.J. Connors et al. / Journal of Structural Biology 177 (2012) 314–328

Species were identified as T. marmorea and distinguished from 2.4. 3D-printing Tonicella rubra by the height/width aspect ratio of the fourth plate (0.44 vs. 0.29), and appearance of the girdle (leathery to the A scaled-up macroscopic prototype of the chiton shell plate naked eye vs. densely clothed with club-shaped, calcareous cor- assembly was fabricated using a three-dimensional printer (3DP, puscles), respectively (Kaas and Van Belle, 1985). Macroscopic ZPrinterÒ 310 Plus, ZCorporation, USA) using the stereolithography morphological chiton terminology used was defined and/or stan- (STL) files that were created from the micro-computed tomogra- dardized by Schwabe (2010) while chiton shell plate layer nomen- phy data. The material used was a plaster powder (ZPÒ 131 pow- clature used was derived from Bergenhayn (1930) and Laghi and der, ZCorporation, USA). Eighty-nine micrometer thick layers Russo (1978, 1980). Molluscan microstructure terminology used were laid down using a commercially available binder (ZCorpora- was defined by Taylor and Layman (1972) and Carter and Clark tion, USA) at a vertical build speed of 25 mm/h. After printing, (1985). the prototype was immersed in a wax bath to ensure a smooth surface. 2.2. Micro-computed tomography (microCT) 2.5. Optical and scanning electron microscopy (SEM) The complete shells (plates 1–8 and girdle) of T. marmorea were scanned with a micro-computed tomography system (lCT40, For cross-sectional imaging, plates were embedded in a fast Scanco Medical AG, Switzerland) operated at 70 kV and 114 lA. cure epoxy (Loctite, USA) and sectioned with a diamond impreg- Microtomographic slices were recorded every 10 lm and were nated saw (Buehler Isomet 5000, Lake Bluff, IL) at 875 rpm. Embed- reconstructed with 10 Â 10 lm voxels in plane. For the chiton pre- ded samples were polished stepwise with aluminum oxide pads sented in Fig. 1a, the 3D information of the plate assembly was par- with roughness varying from 9 to 0.1 lm (South Bay Technologies, titioned into contributions from each individual plate using the CA) and then with 500 nm silica nanoparticles on microcloth threshold and contour functions of Scanco MicroCT Software. For (Buehler, IL). Some samples were slightly etched with 0.5 M EDTA the chiton presented in Fig. 1b, the 3D information of the plate for 5 min to improve contrast between microstructurally distinct assembly was segmented using the threshold and region growth layers. Additionally, some plates were cryofractured by immersing functions of image processing software package Mimics (Material- them in liquid nitrogen for 5 min and then breaking them with a ise, Belgium). Mimics was also used to create all three-dimensional hammer. Optical images were taken with a Nikon ECLIPSE LV100 images, including those with transparency effects. The three- microscope (Tokyo, Japan). Cross-sectional SEM samples were dimensional geometric information of the plate assemblies was fixed on a steel support using a conductive tape and then sput- converted into three-dimensional polygonal meshes (stereo lithog- ter-coated with 5 nm of gold–palladium in a Denton Vacuum raphy) using Scanco MicroCT Software for the chiton presented in Desk II (Moorestown, NJ). A JEOL JSM 6700 (Peabody, MA) scanning Fig. 1a or Mimics for the chiton presented in Fig. 1b. Meshes were electron microscope was used for imaging at an acceleration volt- reduced in size as needed using the smooth, reduce, and remesh age 10 kV. Demineralized SEM samples were secured in a holder, tools of 3-matic (Materialise, Belgium). Cross-sectional images covered with carbon for 1 min using an Edwards S150B sputter were created from the surface meshes using the cut function of coater (West Sussex, United Kingdom) and imaged with an ESEM the simulation module of Mimics. Open source image analysis soft- XL 30 Philips (Amsterdam, Netherlands) or LEO DSM 982 Gemini ware ImageJ (Rasband, 1997–2011) was used to measure the (Oberkochen, Germany) scanning electron microscope. dimensions of cross-sectional images. The spatial distribution of thickness of each individual plate and of the entire plate assembly 2.6. Thermogravimetric analysis (TGA) (Fig. 3a and b, respectively) of the chiton presented in Fig. 1a was calculated using a spherical method in which each point in 3D Plates were lightly ground with a pestle and mortar. Samples space is assigned a thickness value corresponding to the diameter were then vacuum dried at 110 °C overnight to remove residual of the largest sphere centered on that point which can fit within water. TGA was carried out from 110 to 500 °C at 2.5 °C/min on a the boundaries of the plate (Bouxsein et al., 2010). TA Instruments TGA Q50 (New Castle, DE). Weight loss due to com- bustion of organic material was attributed to the temperature 2.3. Calculations of plate-to-plate overlap and curvature range 110–475 °C(Zaremba et al., 1998; Neves and Mano, 2005).

In Fig. 2f, overlap was quantized using aerial projections (in the 2.7. Mercury porosimetry ‘‘y’’–‘‘z’’ plane) of the plates and plate-to-plate overlapping regions of Fig. 2d and e. For each intermediate plate, total overlap percent- The volume percent porosity of three plates (2–4) was mea- age was calculated by summing the projected areas of the two sured by a mercury porosimeter (Autopore IV 9500, Micromeritics, overlapping regions, and expressing the sum as a percentage of USA), which operated at pressures between 3.7 kPa and 14 MPa, the projected area of the entire plate which encompasses them. corresponding to pore diameters of 404 and 0.107 lm, respec- For the first and eighth plates, total overlap percentage was calcu- tively. A standard contact angle of 140°, which was used previously lated by expressing the projected area of the single overlapping re- by Gane et al. (2004) to study the porosity of calcium carbonate gion (the overlapping region of plates 1 and 2, and plates 7 and 8, structures, was assumed in the pore size calculations. respectively) as a percentage of the projected area of the respective plate. In Fig. 5c, overlap was calculated by projecting the overlap- 2.8. X-ray diffraction (XRD) ping regions of plates 3 and 5 onto the long axis (‘‘l’’) of plate 4, summing the projected lengths, and expressing the sum as a per- The mineral phase of the plates was verified using XRD (Philips centage of the cross-sectional length of plate 4. The transverse radii PANalytical X’ Pert PRO diffractometer with CuKa radiation, Neth- of curvature of individual plates and the longitudinal radii of cur- erlands), operating at 45 kV and 40 mA between 10° and 70° (2h). vature of the plate assemblies were calculated using open source ‘‘Circle Fit (Pratt Method)’’ MatLab (MathWorks, MA) code 2.9. Chitin isolation and staining (http://www.mathworks.com/matlabcentral/fileexchange/22643) written by Nikolai Chernov based on an algorithm developed by The organic matrix of chiton plates was isolated by decalcifica- Pratt (1987). tion using 3 M HCl solution as well as a 7.4 pH Osteosoft™ (Merck) M.J. Connors et al. / Journal of Structural Biology 177 (2012) 314–328 317

(a) Dorsal View Anterior (b) Ventral View

Slits

z z

x • y Posterior y x x x L1 (c) z

(e)

y x• z

y T1 (d)

Dorsal 0.5 cm

x • z

Ventral

Posterior Anterior

(f) 35 100

30 Posterior Anterior

) 80 2 25 Plate Area (Fig. 2d) 20 60 Plate Area (Fig. 2e) 15 40 Plate-Plate Overlap Area (Fig. 2d)

10 Plate-Plate Overlap Area (Fig. 2e) Percentage Overlap Percentage Projected Area (mm 20 5 % Total Overlap (Fig. 2d) % Total Overlap (Fig. 2e) 0 0 12345678 Plate Number

Fig.2. microCT images of the three-dimensional structure of the armor plate assembly of Tonicella marmorea; (a–d) correspond to the chiton of Fig. 1a while (e) corresponds to the chiton of Fig. 1b; (a) dorsal view displaying position of cross sections L1 and T1; (b) ventral view; (c) side/ventral view; (d) side view; (e) side view displaying position of cross section T1; (a, b, d and e) include complimentary images created using a transparency effect; (f) projected area in the ‘‘y’’–‘‘z’’ plane (open circle and square symbols) and total overlap percentage (filled circle and square symbols) vs. plate number calculated from the plates and plate-to-plate overlapping regions of the transparent images of (d) and (e). Aerial overlap contributions from all adjacent plates were considered in calculations of total overlap percentage. 318 M.J. Connors et al. / Journal of Structural Biology 177 (2012) 314–328 solution at room temperature. The procedure was monitored using transparent image of Fig. 2d, in the smaller plate assembly with stereo, light and fluorescence microscopy (BZ-8000, Keyonce, lower longitudinal curvature (about the ‘‘x’’-axis), plate-to-plate Japan). Immersion of the isolated organic matrix, inter-plate mate- overlap is not limited solely to the apophyses; a portion of each rial, and girdle in 2.5 M NaOH at 37 °C led to an immediate loss of plate’s jugal area (between the sinus, see Supplementary material brownish pigment as well as of proteins from these specimens. The 1a) is also involved in overlap. In contrast, overlap in the jugal fibrous colorless materials were then washed with distilled water areas of each plate is absent or greatly reduced in the transparent five times, dialyzed against deionized water on Roth (Germany) side profile of the larger, defensively postured plate assembly with membranes with a MWCO of 14 kDa for 48 h at 4 °C, and finally the greater longitudinal curvature (Fig. 2e). In the plate assembly dried at room temperature. Calcofluor White (Fluorescent Bright- with the lower longitudinal curvature (Fig. 2d), the total plate ener M2R, Sigma) was used to confirm the presence of chitin. overlap percentage of the intermediate plates increases from Samples were placed in 0.1 M Tris–HCl at pH 8.5 for 30 min, 52% in plate 2 to 65% in plate 7 (Fig. 2f). In contrast, in the plate stained using 0.1% Calcofluor White solution for 30 min in dark- assembly with the greater longitudinal curvature (Fig. 2e) the total ness, rinsed five times with deionized water, dried at room temper- overlap percentage of the intermediate plates increases from 44% ature, and finally observed using fluorescence microscopy (Ehrlich in plate 2 to 58% in plate 7. The average total plate overlap per- et al., 2007). centages of the two plate assemblies presented in Fig. 2d and e are 62.27 ± 11.45 and 48.00 ± 9.07, respectively. 2.10. Fourier-transform infrared spectroscopy (FTIR) The thickness distribution of the plate assembly of the smaller chiton presented in Fig. 2d(Fig. 3a) shows that the intermediate Infrared spectra were recorded on a Nicolet 210 FT-IR Spec- plates have a similar spatial distribution of thickness, in which trometer. Two hundred and fifty scans were recorded at a spectral the central non-overlapping regions are thickened (0.85 mm) rel- resolution of 2 cmÀ1. All spectra were baseline corrected with a ative to the anterior and posterior overlapping plate edges two-point linear baseline at 845 and 1890 cmÀ1.Ana-chitin stan- (0.4 mm) and apophyses (0.2 mm). The thin (green) and thick dard from the demosponge Ianthella basta (Brunner et al., 2009) (red) regions of the plate assembly in Fig. 3b mirror the overlap- was used as a control. ping and non-overlapping regions, respectively, of the transparent image of the plate assembly in Fig. 2a. The eight plates of the assembly also have both similar average thicknesses (411 ± 31) 2.11. Amino acid analysis (AAA) and average standard deviation of thickness (222 ± 23) (Fig. 3c). Although thickness is relatively constant across the assembly, Dry, cleaned plates were ground with a pestle and mortar and there is an asymmetric trend in plate volume (Fig. 3c); the head weighed. The fine powder was suspended in deionized water. In plate is 40% larger than the tail plate by volume. Additional struc- a demineralization step, stoichiometric amounts of 1 M HCl were tural data is available in Supplementary material 1, which also la- added dropwise to the solution over the course of 2 days (Treves bels the anatomy of individual plates and defines structural et al., 2003). After complete demineralization, the solution was parameters. Visualization of plate morphology and imbrication is centrifuged (3000 rpm, 10 min, Eppendorf Centrifuge 5804 R) to facilitated using a macroscopic prototype of the plate assembly separate soluble material from the insoluble, precipitated material. (Supplementary material 2). The soluble material was dialyzed exhaustively (Spectra Pro 7 dial- Fig. 4a and b show the L1 cross sections of the plate assemblies ysis membranes, 3500 MWCO, part # 132112) to remove salts and of the chitons presented in Fig. 1a and b, respectively. L1 bisects small molecules. The insoluble material was lyophilized and dried the jugal sinus of each plate 2–8, so the apophyses are absent. In overnight in a vacuum oven at 50 °C. Samples were hydrolyzed for the plate assembly with lower longitudinal curvature (Fig. 4a), AAA, which was performed using a Waters system with Breeze the heterogeneous cross-sectional plate geometry and plate-to- software by Bio-Synthesis, Inc. (Lewisville, TX). plate overlap result in a relatively constant thickness and approximately continuous convex curvature of the plate assembly as a 3. Results whole. To approximate their longitudinal (about the ‘‘x’’-axis) radii of curvature, 16 landmark points were selected for each assembly 3.1. Three-dimensional structure and geometry of the plate assembly from the two end points of each of the eight plates along their lon- of T. marmorea gitudinal axes. As illustrated in Fig. 4c and d, circles were fit to these points using a direct least squares fitting algorithm (Pratt, The three-dimensional structure of the plate assemblies of two 1987). The longitudinal radii of curvature of the L1 cross sections representative T. marmorea chitons (Fig. 1a and b) were derived of Fig. 4a and b are 9.15 and 7.39, respectively. The transverse from renderings of microCT data. In their curved states, the plate (about the ‘‘z’’-axis) radii of curvature of the fourth plates of each assemblies of the chitons seen in Fig. 1a and b have dimensions assembly were approximated using the same method. Eleven land- 1.7 Â 0.95 Â 0.45 cm and 1.66 Â 1.4 Â 1.38 cm (length (‘‘z’’- mark points from cross section T1 were used in the calculation for direction) Â width (‘‘x’’-direction) Â height (‘‘y’’-direction)), respec- each plate: the midpoint of cross section L1, the midpoints of cross tively. The girdle, as well as the inter-plate material (transverse sections L2–L5 (left side) and their analogous midpoints on the muscle and cuticular layer), were very slightly or non-mineralized right side, and left and right base points of the insertion plates and consequently did not absorb X-rays well enough to be clearly (Fig. 5a and b). The transverse radii of curvature of the T1 cross sec- visible in the microCT scans. Fig. 2a and b display dorsal and ven- tions of the fourth plates seen in Fig. 5a and b are 4.60 and 6.71, tral views, respectively, of the plate assembly of the smaller chiton respectively. To compare the curvature of the two plate assemblies of Fig. 1a along with complimentary transparent images highlight- and account for the size disparity, two curvature indices were de- ing plate-to-plate overlap (darkened regions). The broad ‘‘u’’ shape fined (Fig. 4f). The first index, the longitudinal curvature index of the overlapping regions results from each plate’s (except the (L.C.I), is defined as the length of plate 4 divided by the longitudinal first plate) two anterior projections, the apophyses, which imbri- radius of curvature of the plate assembly in cross section L1. This cate with adjacent anterior plates and are separated by a sinus. yields L.C.I. values of 0.43 and 0.70 for the plate assemblies of Side views of the plate assemblies are shown in Fig. 2d and e, Fig. 4a and b, respectively. The second index, the transverse curva- and are accompanied by transparent images. As illustrated by the ture index (T.C.I), is defined as the width of plate 4 divided by its M.J. Connors et al. / Journal of Structural Biology 177 (2012) 314–328 319

(a) 8 Posterior

(b)

6 3

y 5 • x

Anterior 3 mm z

AP JS AP 0 mm 0.94

Total Volume (c) Average Thickness 25 1 3 20 0.8 ) 3 15 0.6

2 10 0.4 Volume (mm Volume Thickness (mm) Thickness

5 0.2

0 0 12345678 1 Plate Number

Fig.3. Thickness distribution of the armor plate assembly of Tonicella marmorea presented in Fig. 1a; (a) dorsal view of the spatial distribution of thickness for each plate 1–8; (b) dorsal view of the spatial distribution of thickness of the entire plate assembly; (c) total volume (triangle symbols) and average thickness (circle symbols) as a function of plate number. Error bars in (c) refer to standard deviations of the dataset. Terminology: AP, apophyses; JS, jugal sinus. transverse radius of curvature in cross section T1. This yields T.C.I of gap ‘‘n’’ was measured along the long axis of plate number values of 0.48 and 0.50 for the fourth plates of Fig. 5a and b, ‘‘n + 1’’ in cross section L1. For example, the separation distance be- respectively. tween plates 4 and 5 (Gap 4) was measured along the long axis Fig. 4e shows how the inter-plate separation distance changes (‘‘l’’) of plate 5 (Fig. 4b). To account for the size disparity between as a function of gap number for the two plate assemblies seen in the two assemblies, the separation distances were normalized by Fig. 4a and b. Gap number ‘‘n’’ corresponds to the gap between dividing each one on by the length of the fourth plate of the plate number ‘‘n’’ and ‘‘n + 1’’. The inter-plate separation distance respective assembly. As seen in Fig. 4e, the normalized separation 320 M.J. Connors et al. / Journal of Structural Biology 177 (2012) 314–328

P4 (a) L1 (b) L1 P5 2 mm P3 ℓ x • z Dorsal P6 P2 y Ventral

P7 P1 Posterior Anterior P8

L. Radius = 7.39 mm

L. Radius = 9.15 mm

(e) 0.30

0.24 (f) Fig. 6a Fig. 6b 0.18 L.C.I 0.43 0.70 Plate Separation Separation Plate - 0.12 T.C.I 0.48 0.50 (Fig. 6a) 0.06 (Fig. 6b) R/t (longitudinal) 10.73 6.44

Normalized Inter Normalized 0.00 1234567 R/t (transverse) 5.39 5.84 Gap Number

Fig.4. L1 (longitudinal bisector) cross-sectional structure of the plate assembly of T. marmorea; (a) cross section L1 of plate assembly of the chiton presented Fig. 1a; (b) cross section L1 of plate assembly the chiton presented in Fig. 1b; (c and d) circles ﬁt to 16 landmark points (the two end points of each of the eight plates along their longitudinal axes) from (a) and (b) using a direct least squares ﬁtting algorithm (Pratt, 1987); (e) normalized inter-plate separation distance vs. gap number. Gap number ‘‘n’’ corresponds to the gap between plate number ‘‘n’’ and ‘‘n + 1’’. The inter-plate separation distance of gap ‘‘n’’ was measured along the long axis of plate number ‘‘n + 1’’ in cross section L1 and normalized by dividing by the length of the fourth plate of the respective assembly; (f) curvature indices and shell thickness ratios. The longitudinal curvature index (L.C.I) is calculated by dividing the length of plate 4 by the longitudinal radius of curvature of the plate assembly in cross section L1. The transverse curvature index (T.C.I) is calculated by dividing the width of plate 4 by its transverse radius of curvature in cross section T1. Shell thickness ratios were calculated by dividing the appropriate radius of curvature by the average of the maximum thicknesses of the fourth plates in cross sections of L1–L5 (Fig. 5a and b).

distances of gaps 1 and 2 are similar in both plate assemblies. gaps 5 and 6 are also larger in the plate assembly with the larger However, the normalized separation distances of gaps 3, 4, and 7 L.C.I., although the increase is not as pronounced. In both plate are 75%, 100%, and 200% greater, respectively, in the plate assemblies, the trend of normalized separation distance vs. gap assembly with a L.C.I. of 0.70 (Fig. 4b) relative to the plate assembly number is asymmetric; the separation distance of gap 2 is 160% with a L.C.I of 0.43 (Fig. 4a). The normalized separation distance of larger than that of gap 6. M.J. Connors et al. / Journal of Structural Biology 177 (2012) 314–328 321

L1 L2 • T1 L3 (a) L1 T1 z (b) • • L2 • • x • • L3 • • L4 L4 • • y • • • • L5 L5 • • k • • k • P5 T. Radius = 4.6 mm • • T. Radius = 6.71 mm • P4 P4 P5 Dorsal P3 P4 P4 L1 L1 P5 2 mm VTC Ventral P3

L2 P5

ℓ L2 L3

L5 L3 AP

% Total Overlap (Fig. 7a) % Total Overlap (Fig. 7b) (c) Max. Thickness (Fig. 7a) Max. Thickness (Fig. 7b) 100 1.50 L4

80 1.25

60 1.00 L5 40

% Total Overlap Total % 0.75

20 Thickness Max. (mm) Posterior Anterior 0 0.50 12345 Cross Section (L)

Fig.5. Cross-sectional structure of the plate assembly of T. marmorea; (a and b top) T1 cross sections of the fourth plate 4 of the plate assemblies of the chitons presented in Fig. 1a and b, respectively. Black circles indicate the eleven points used to calculate the transverse radius of curvature of the fourth plate of each plate assembly using a direct least squares ﬁtting algorithm (Pratt, 1987); (a and b bottom) Morphological changes of longitudinal cross sections (L1–L5) of the fourth plate of each plate assembly. Cross sections L1–L5 were taken at 20% increments along line segment ‘‘k’’; (c) total overlap percentage and maximum thickness of plate 4 vs. longitudinal cross section number. Total overlap percentage was calculated by projecting the overlapping regions of plates 3 and 5 onto the long axis (‘‘l’’) of plate 4, summing the projected lengths, and expressing the sum as a percentage of the cross-sectional length of plate 4. Maximum cross-sectional thickness was measured perpendicular to the long axis (‘‘l’’) of plate 4. Terminology: AP, apophyses; VTC, ventral tegmental callus.

Fig. 5a and b (top) show the dimensions and geometry of trans- the midpoint of cross section L1 to the eave of the tegmentum be- verse cross section T1 of the two plate assemblies. T1 was taken in low. A similar line segment was named ‘‘k’’ by Bergenhayn (1930 the region in which plates four and five overlap, and thus includes Fig. 1, page 14), and we adopt this notation (Fig. 5a). Fig. 5c dis- both plates. Longitudinal cross sections L2–L5 were taken at 20% plays how the total overlap percentage of a representative inter- increments along the line segment which runs diagonally from mediate plate (number four) evolves laterally from L1 to L5. In 322 M.J. Connors et al. / Journal of Structural Biology 177 (2012) 314–328 the both plate assemblies, overlap is heterogeneous in nature, but columns possess a ‘‘wavy’’ surface topography, which has a wave- the heterogeneity is more pronounced in the plate assembly with length approximately equal to their width (0.3 lm) (Fig. 7i). The the higher L.C.I. The total overlap percentage of plate four of the periodic surface elevations on one side of the interface align with plate assembly with the lower L.C.I. is 60% in cross sections L1 surface depressions on the opposite side. Layers 4 and 5, the ante- and L2, and 82.5% in cross sections L3–L5. In contrast, the total rior and posterior myostracum, respectively, both have a fine- overlap percentage of plate four of the plate assembly with the grained homogeneous microstructure with grains approximately higher L.C.I. is 0% in cross sections L1 and L2, and increases 1 lm in diameter (Fig. 7e and j). The anterior myostracum stems approximately linearly from 50% to 90% across cross sections from the impressions of the oblique muscles while the posterior L3–L5. The average total overlap percentages of the fourth plates myostracum lies in the impression of the transverse muscle. of the plate assemblies with L.C.I. of 0.43 and 0.70 are 73.9% and Together, the articulamentum, mesostracum, and anterior myos- 44.6%, respectively. tracum form the apophyses (Fig. 6a). The bottom layer, the hypostracum, has a simple crossed-lamellar microstructure identical to 3.2. Multilayered microstructure of the intermediate plates of T. that of mesostracum, except that the long axes of the first order marmorea lamellae are oriented perpendicular to the ventral plate surface (Fig. 7f and k). This was supported by XRD of the ventral shell sur- The intermediate plates of T. marmorea are composed of six ara- face, which found two preferred grain orientations corresponding gonite-based layers (Fig. 6). The dorsal mesostracm was not ob- to the two lamellar dip directions (Fig. 8a, top dotted line). First or- served in any plates used in this study, and so we refer to the der columns often slightly deviate from the perpendicular orienta- ventral mesostracum simply as the mesostracum. Fig. 6a and b tion, become thinner and disappear, or become thicker and branch. contain representative longitudinal (L4) and transverse (T1) The hypostracums’s transverse distribution of thickness is inverse cross-sections, which are each accompanied by a schematic dia- to that of the articulamentum; it is approximately 0.9 mm thick gram that illustrates the spatial distribution of the six layers. The in the jugal area and absent laterally, at the start of the insertion key to the schematics (Fig. 6d) relates textures and colors to micro- plates (Fig. 6b). Longitudinally, the hypostracum composes the structures and layers. bulk of the central callus and is absent from the apophyses and The outermost layer, the tegmentum, has a homogeneous thick- insertion plates. ness of 155 lm and is infiltrated with the aesthete canal system (Fig. 6a), which constitutes 7.5% of each plate by volume as measured by mercury porosimetry (Supplementary material 1i). Main 3.3. Chemical composition of the intra-plate organic matrix, girdle, and aesthete channels in the tegmentum run roughly longitudinally, inter-plate material of T. marmorea are separated by 20 lm, and have a diameter of 40 lm, which lies in the lower end of the range (40–75 lm) found across genus The intermediate plates of T. marmorea have an organic matrix Tonicella (Vendrasco et al., 2008). Scanning electron microscopy re- content of 2.6 wt.% (aragonite content of 97.4 wt.%), as mea- vealed that the tegmentum has a fine-grained homogeneous sured by thermogravimetric analysis (Supplementary material microstructure (Fig. 7b and g). XRD of the dorsal shell surface 1i). The organic matrix, which was obtained after a 24 h gentle found no preferred grain orientations (Fig. 8a). Grains are approx- demineralization using Osteosoft™ solution at room temperature, imately 1 lm in diameter, but are slightly elongated along one axis showed a strong resistance to alkali treatment (Supplementary ventrally, below the main aesthete channels. Posteriorly, the teg- material 3a). It remained stable after submersion in 2.5 M NaOH mentum folds underneath the plate and becomes the posterior at 37 °C for 30 days. The girdle and inter-plate material, which is myostracum (layer 5), which lies in the impression of the trans- composed of the transverse muscle and cuticular layer, displayed verse muscle (Fig. 6a). The transverse muscle, which runs longitu- similar properties. After alkali dissolution of their proteinaceous dinally from the eave of the tegmentum to the tip of the apophyses, components, Calcofluor White staining (Supplementary material fills the gap between overlapping plates (Fig. 6c). The thickness of 3b) and FT-IR spectroscopy indicated the presence of a-chitin the transverse muscle ranges from 100 to 250 lm. Slit ray and within the shell plate organic matrix, girdle, and inter-plate mate- jugal aesthete channels that begin in the tegmentum, pass though rial (Fig. 8b). Amino acid analysis of the intra-plate organic matrix the lower layers, and exit on the ventral shell surface are insulated revealed that the total amount of protein in the shell plates is by a fine-grained homogeneous layer that is a couple of microns 0.17 wt.% (Supplementary material 4b). thick. The second layer, the articulamentum, has a composite prismatic microstructure (Fig. 7c and h). Bundles of prisms fan out dorsally and ventrally from the center of the layer. The length of the 4. Discussion prism bundles ranges from approximately 3–10 lm. Individual prisms are rectilinear in shape and have dimensions 0.3 Â 0.3 lm In segmented natural exoskeleton systems, there exists an (width Â height). The thickness of the articulamentum ranges from interplay between the ‘‘inherent materiality’’ of the individual ar- 0 mm in the jugal area to approximately 0.9 mm laterally, at the mor sub-units (i.e. microstructure, multilayering, anisotropy, grad- start of the insertion plates (Fig. 6b). ing, etc.) and the larger length scale morphometric or geometric The articulamentum is followed by the mesostracum, which has design to simultaneously achieve the necessary protection and a simple crossed-lamellar microstructure (layer 3) (Fig. 7d and i). It biomechanical flexibility/mobility. This interplay can vary dramat- branches off from the hypostracum near the plates’ body diagonal, ically among different systems. Here, we explore this topic in a along which the slit ray aesthete channels run (Fig. 6a). The long model system of the chiton T. marmorea by using a suite of exper- axes of first order lamellae are oriented at a 45° angle relative imental techniques to quantify critical design aspects such as (from to the ventral plate surface and have variable width between 1 smallest to largest length scale): (1) the microstructure, multilay- and 2 lm. The layer lacks well-defined second order lamellae. ering and material properties of the individual armor sub-units, Third order lamellae are rectilinear in shape and have dimensions (2) the geometry of the individual armor sub-units, (3) the 15 Â 0.3 Â 0.3 lm (length Â width Â height); each first order col- interconnections between armor sub-units, (4) the degree and umn is 3–6 third order lamellae wide. The angle between bundles type of armor unit overlap, and the interaction and correlation of third order lamellae in adjacent first order columns is 40°. between these design aspects. We will discuss each of these Third order lamellae on the interface between adjacent first order aspects following. M.J. Connors et al. / Journal of Structural Biology 177 (2012) 314–328 323

(a) Dorsal 1 L4

SR 1 2 2 4 3 6

Anterior Ventral Posterior 5 Transverse Muscle 0.5 mm Oblique Muscle

1 (b) 6 T1 (c) Transverse z SL Muscle x x

y 2 3 AP 4 2 mm 0.5 mm IP IP

(d) Layer Name Microstructure Symbol(s) 1 Tegmentum Granular 2 Articulamentum Composite Prismatic 3 Mesostracum Crossed-Lamellar a) b) 4 Ant. Myostracum Granular 5 Post. Myostracum Granular 6 Hypostracum Crossed-Lamellar a) b) - Muscle Tissue -

Fig.6. Cross-sectional structure of intermediate plates of Tonicella marmorea by dark-ﬁeld optical microscopy; (a) composite image of polished longitudinal cross-section (L4) and complimentary schematic; (b) composite image of polished transverse cross-section (T1) and complimentary schematic; (c) longitudinal cross-section displaying transverse muscle between two adjacent intermediate plates; (d) table relating textures in schematics to layers and microstructures. Terminology: AP, apophyses; IP, insertion plate; SR, slit ray.

In order to understand functional design, it is important to con- the sea star Leptasterias littoralis in shallow water (less than 6 m) sider the environment and types of predatory attacks against for and the wrasse Tautogolabrus adspersus and winter flounder Pseu- which specific natural armor systems are designed. Langer dopleuronectes americanus at greater depths; secondary predators (1978) observed that T. marmorea from the northwestern Atlantic include the crabs Cancer borealis and Cancer irrorarus and the lob- is found most abundantly on subtidal calcareous algae covered ster Homarus americanus. Possible predatory attacks include biting rock, and displays cryptic coloration. Field observations led Langer (fish), pinching with claws (lobsters and crabs), drilling with rad- (1978) to suspect that the primary predators of T. marmorea were ulas (sea snails), inserting the cardiac stomach underneath or 324 M.J. Connors et al. / Journal of Structural Biology 177 (2012) 314–328

(a) Polished & Etched Sample Cryofractured Samples Dorsal L4 (b) (g)

1 µm 1 µm 1- Tegmentum 1- Tegmentum (c) (h)

1 µm 1.5 µm

c (d) (i) 2- Articulamentum 2- Articulamentum

d 3 e 1 µm 1.5 µm 0.3 µm 4 (e) (j)

1 µm 0.5 µm 6 - Hypostracum

(f) (k) Muscle

50 µm Ventral 1 µm 3 µm

Fig.7. Spatial distribution of microstructures of intermediate plates of Tonicella marmorea by SEM; (a) composite image of the boxed (white dashes) region of longitudinal cross section L4 of Fig. 6a, which was polished and etched with 0.5 M EDTA for 5 min to increase image contrast. Images (b–f) correspond to magnified regions of (a) and preserve microstructure orientation with respect to cross section L4. Images (g–k) were obtained from cryofractured samples and display true microstructure dimensions and geometry; (b and g) layer 1, tegmentum, fine-grained homogenous; (c and h) layer 2, articulamentum, composite prismatic; (d and i) layer 3, mesostracum, simple crossed lamellar; (e and j) layer 4, anterior myostracum, fine-grained homogenous; (f and k) layer 6, hypostracum, simple crossed lamellar.

between the shell plates (sea stars), and smashing against rocks observed lining both the dorsal and ventral sides of the articula- (birds). mentum in Chiton olivaceous (Kreusch, 1981;Laghi and Russo, 1978; Poulicek and Kreusch, 1983), Leopidopleurus cajentanus 4.1. Comparison of the shell plate microstructures of T. marmorea with (Laghi and Russo, 1980), Liolophura gainardi (Kreusch, 1981; other Polyplacophora and Mollusca Poulicek and Kreusch, 1983), and Liolophura japonica (Chen, 2010). However, we only observed it lining the articulamentum The mesostracum was originally named by Bergenhayn (1930) ventrally in T. marmorea, and Baxter and Jones (1981) did not ob- for its position between the tegmentum and articulamentum in serve it at all in Lepidochitona cinereus. In addition, Kreusch Acanthopleura spinigera. He observed that it was similar in struc- (1981) observed the mesostracum within the articulamentum in- ture to the hypostracum, which is now recognized as having a sim- stead of lining it dorsoventrally in Cryptochiton stelleri. Laghi and ple crossed-lamellar microstructure. The mesostracum has been Russo (1978, 1980) and Kreusch (1981) note that in species which M.J. Connors et al. / Journal of Structural Biology 177 (2012) 314–328 325

(a) species that contain the dorsal mesostracum may be regarded as having seven, or possibly eight. Laghi and Russo (1978, 1980), Kreusch (1981), and Poulicek and Kreusch (1983) refer to the dorsal and ventral mesostracum layers collectively as the pallial myos- Ventral tracum. With the sole exception of the dorsal mesostracum, the (102) (112) Surface spatial distribution of microstructures in T. marmorea, C. olivaceous (see longitudinal and transverse cross-sectional schematics in Dorsal Figs. 2 and 4, Laghi and Russo, 1978), and L. gainardi (see longitu- Surface dinal schematic in Fig. 12, Kreusch, 1981) are very similar. Poulicek Aragonite and Kreusch (1983) note that in the evolutionary series of chitons, the tegmentum becomes thinner (disappearing almost completely (111) in the more evolved Cryptochiton), the articulamentum becomes Intensity thicker, and the hypostracum, mesostracum, and myostracal layers show little variation in proportions. Similar orientation, thickness, and branching irregularities of the first order lamellae of the crossed-lamellar layers of the shell plates of T. marmorea have also been reported in the shells of other mollusks (Currey and Kohn, 1976; Taylor and Reid, 1990; Wilmot et al., 1992). The width of first order lamellae in the crossed-lamellar layers of the shell plates of T. marmorea (1–2 lm), Acanthople- ura brevispinosa (1.7–5 lm) (Wilmot et al., 1992), Chiton olivaceus 25 30 35 40 45 50 55 (2–3 lm) (Laghi and Russo, 1978), Lepidopleurus cajetanus Two-Theta (deg.) (3 lm) (Laghi and Russo, 1980), Liolophura japonica (3 lm) (Chen, 2010), as well as the range found in 36 chiton species rep- (b) α-Chitin standard from I. basta resenting 19 genera (3–5 lm) (Haas, 1972) lies in the extreme lower end of the range (1–40 lm) found in crossed-lamellar Organic Matrix after NaOH Treatment microstructures across Mollusca (Carter and Clark, 1985; Wilmot Native Inter-Plate Material et al., 1992). As previously observed by Haas (1976, 1981), Poulicek and Kreusch (1983), and Carter and Hall (1990), the simple Inter-Plate Material after NaOH Treatment crossed-lamellar structures present in chiton shell plates are de- Native Girdle void of well-defined second order lamellae. The width of third or- Girdle after NaOH Treatment der lamellae found in the crossed-lamellar layers of T. marmorea, Chiton olivaceus (Laghi and Russo, 1978), and Lepidopleurus cajetanus (Laghi and Russo, 1980) are identical (300 nm), and similar to the range (200–300 nm) observed by Haas (1972). The angle be- 1008 1061 tween the two dip directions of third order lamellae in adjacent columns of first order lamellae in T. marmorea is approximately 1151 1628 1107 1543 1374 947 890 40°, which is similar to the dip angles previously observed in Chi- ton olivaceus (50°)(Laghi and Russo, 1978), Lepidopleurus cajetanus (45°)(Laghi and Russo, 1980), and Acanthopleura brevispinosa (32–45°)(Wilmot et al., 1992), and much less than the range (60–85°) observed by Haas (1972).

4.2. Chemical composition of the shell plate organic matrix of T. Intensity marmorea

The intermediate plates of T. marmorea were determined to have an organic matrix content 2.6 wt.% by TGA. Poulicek and Kreusch (1983) found that the shell plate organic matrix content ranged from 0.48 to 1.55 wt.% (greatest in Tonicella lineata)in11 species; however, they only considered the insoluble portion of the matrix in their measurements. In contrast to the proteinaceous composition of organic matrices of other mollusk shells, those of chiton shell plates possess a very large amount of the aminopoly- 1650 1450 1250 1050 850 650 saccharide chitin (Furuhashi et al., 2009). An earlier study found Wavenumber (cm-1) the range of the weight percent of chitin in the insoluble portion of the shell plate organic matrix, girdle, and organic matrix of the Fig.8. Chemical composition of intermediate plates of Tonicella marmorea; (a) X-ray mineralized girdle spicules to be 16–41, 1.6–16, and 0.2–15, diffraction pattern of the dorsal and ventral surfaces of an intermediate plate; (b) respectively (Poulicek and Kreusch, 1983). In addition, the shell infrared spectra of the intra-plate organic matrix, girdle, and inter-plate material before and after NaOH treatment to remove proteins. plate organic matrix of Acanthopleura villantii was found to contain a chitin/protein ratio of 6.9, which is over a hundred times larger than that found in shells of other mollusks, speciﬁcally bivalves (Furuhashi et al., 2009). Our results are consistent with these stud- contain the dorsal mesostracum, it is split dorsoventrally into two ies. Because of its resistance to alkali treatment, it is very simple to sub-layers by a row of aesthete channels. Thus, while the interme- isolate chitinous material from most composite biomaterials, diate plates of T. marmorea have six layers, those of other chiton including cases where chitin is bound to proteins or pigments 326 M.J. Connors et al. / Journal of Structural Biology 177 (2012) 314–328

(Ehrlich, 2010a; Ehrlich et al., 2010). After alkali treatment, the for a large portion of the energy dissipated during fracture of the shell plate organic matrix, girdle, and inter-plate material of T. shell of Strombus gigas (Kamat et al., 2000; Kamat et al., 2004). marmorea displayed FTIR spectra very similar to an a-chitin stan- The aforementioned surface waviness of the third order lamellae dard from the demosponge I. basta (Fig. 8b). The protein content may generate a strain hardening mechanism similar to that found (0.17 wt.%) of the shell plates of T. marmorea is similar to the range in nacre, in which the ‘‘dovetails’’ of individual nacre tablets pro- found in the composite prismatic spines and scales (0.07– duce a progressive tablet locking in tension that is responsible 0.23 wt.%) of Acanthopleura vaillantii, Acanthopleura spinigera, for a strain at failure (1%) that is an order of magnitude greater Nuttalina fluxa, and Ischnochitonina sp. (Treves et al., 2003). This than that of non-biogenic aragonite (Barthelat et al., 2007). is consistent with results of Taylor and Layman (1972), who found The transverse thickness distribution (900 lm thick laterally that non-nacreous microstructures including crossed-lamellar, and decreasing to zero centrally towards the jugal area, Fig. 8b) composite prismatic, and homogeneous have a protein content less of the composite prismatic articulamentum, suggests that it func- than 0.4 wt.%. The amino acid profile of the shell plate organic ma- tions to resist circumferential (roughly longitudinal, parallel to trix of T. marmorea (Supplementary material 4) is consistent with transverse ‘‘z’’-axis in Fig. 8b) tensile stress and to provide a stiff, those of Chiton marmoratus and Acanthopleura granulata (Haas, hard layer to resist side penetration and pinching. While the prism 1972), with the sole exception of glycine content, which is about bundles in the articulamentum are aligned parallel to interfaces of 8 mol% greater in C. marmoratus and A. granulata. Calcification of adjacent layers (the tegmentum dorsally and mesostracum ven- the shell plates is likely controlled by the templating activity of trally) in the middle of the articulamentum, they are aligned acidic proteins (Ehrlich, 2010b), which may be attached to the chi- approximately perpendicular to theses interfaces at the layer junc- tinous network of the organic matrix (Supplementary material 3c). tions. This likely serves to provide discrete inter-prism structural pathways for cracks to propagate ventrally for easy arrest by the 4.3. The biomechanical role of the inherent materiality of the crossed-lamellar layers (Wang et al., 2009). The inner articulamen- individual armor sub-units tum and hypostracum layers may also function to resist drilling attacks. Chiton tuberculatus has been observed alive with drill holes The multilayered structure of the individual armor sub-units that bore through the tegementum, but left the inner layers intact plays a significant role in direct penetration resistance in complex (Arey and Crozier, 1919). multiaxial loading configurations by predators (Bruet et al., 2008; In contrast to the inner shell plate layers, the outer tegmen- Wang et al., 2009). The sequence and spatial heterogeneity of the tum’s primary role is likely sensory rather than mechanical in nat- layers of the intermediate plates of T. marmorea suggests a struc- ure; the aesthetes, a complex sensory network of tissue-filled tural response to different loading conditions experienced by dif- channels, are embedded in the tegmentum. In a secondary ferent regions of the plates. The transverse distribution of mechanical role, the tegmentum may operate as a brittle outer crossed-lamellar hypostracum (900 lm thick in the jugal area shield, in which the main aesthete channels act as sacrificial stress and decreasing to zero laterally, Fig. 8b) and orientation of first or- concentrators that distributes a concentrated load (e.g. biting at- der lamellae (long axes perpendicular to the ventral plate surface) tack) over a large area of the plate. The homogeneous microstruc- may function to resist transverse bending (about the ‘‘z’’-axis). ture of the tegmentum implies that it may also function to resist Three-point bending tests by Currey and Kohn (1976) on samples dissolution. Walter and Morse (1984) showed that finely crystal- of Conus spp. showed that the flexural strength of the crossed- line microstructures have higher dissolution rates than coarser lamellar microstructure is highly anisotropic; they determined ones, which lead Taylor and Reid (1990) to suspect that coarse the flexural strength to be 70 MPa in the axial direction (perpen- granular or irregular prismatic microstructures are more dicular to long axes of first order lamellae) and 200 MPa in the dissolution resistant than the finely crystalline crossed-lamellar transverse direction (parallel to long axes of first order lamellae). microstructure. As previously proposed by Haas (1981), the homo- Bending in the axial direction can break the layer by simply pulling geneous myostracal layers’ location in the impressions of the late- the first order lamellae apart from each other, while bending in the ro-pedal, oblique, and transverse muscles suggests that the transverse direction cannot break the layer without breaking each homogeneous microstructure may be best suited for muscle first order lamellae across its long axis (Currey and Kohn, 1976). attachment. The bending tendency of the plates will depend on the boundary conditions at the ambitus (Telford, 1985). Possible ‘‘abutment’’ effects of the girdle and concave substratum, and ‘‘tie rod’’ effects of 4.4. Geometric design aspects of the armor sub-units of T. marmorea the muscular system will both increase the bending tendency. Multiple ‘‘channel’’ cracking between first order lamellae is The transverse curvature (curvature of the chiton plates as ob- known to be important to overall damage tolerance of shells with served in the ‘‘x’’–‘‘y’’ plane sections) provide an arch-enhanced inner crossed-lamellar layers (Kuhn-Spearing et al., 1996; Kamat stiffness to resist bending from both top and lateral loading con- et al., 2000), and is likely an important energy dissipation mecha- ditions; this geometry enhancement to mechanical robustness is nism in the shell plates of T. marmorea during longitudinal bending consistent with the thickness distribution of the articulamentum deformation. The structure of the first order lamellae found in the and hypostracum layers. In less curved conformations (e.g. when two crossed-lamellar layers, the inner hypostracum and middle attached to a flat substrate) the longitudinal cross-sectional shape mesostracum, of the shell plates of T. marmorea permits two of each plate provides a curved outer surface that achieves a toughening mechanisms that may generate high channel crack nearly continuous outer surface of the plate-assembly. The densities during fracture: crack bridging by irregular first order cross-sectional shape (centrally thickened) and thickness distribu- lamellae (Kuhn-Spearing et al., 1996), and crack bridging by the tion of each unit imparts an overall spatially uniform thickness roughness (‘‘waviness’’) of the third order lamellae on first order distribution in the plate-assembly. This effect has also been ob- interfaces (Kamat et al., 2004). The lateral area of the plates, which served in other segmented natural armor systems including the likely experiences the largest longitudinal bending deformations, plate assembly of the marine three spine stickleback Gasterosteus contain two middle layers (the articulamentum and mesostracum) aculeatus (Song et al., 2010) and is likely a universal design prin- whose first order interfaces differ in orientation from those of the cipal. Together these features provide the geometric enhancement hypostracum. This arrangement permits crack deflection and and coverage for protection from penetration, bending, and bridging in the middle layers – a mechanism which is responsible pinching attacks. M.J. Connors et al. / Journal of Structural Biology 177 (2012) 314–328 327

4.5. The interconnections between armor units plate assembly. The passive and defensive conformations exhibited differences in longitudinal curvature index (0.43 vs. 0.70), average In contrast to physically interconnected joints observed in some plate-to-plate overlap (62% vs. 48%), cross-sectional overlap other natural exoskeletons (e.g. the peg-and-socket joint of the heterogeneity (60–82.5% vs. 0–90%, fourth plate), and plate-to- scales of Polypterus senegalus (Pearson, 1981), the sliding hinge/ plate separation distance (100% increase in normalized separation ellipsoidal joint of the lateral plates of Gasterosteus aculeatus (Song distance between plates 4 and 5), respectively. In contrast to phys- et al., 2010), the ball-and-socket joint of sea urchin spines (Takah- ically interconnected joints observed in some other natural exo- ashi, 1967)), T. marmorea utilizes an actuating sandwich structure skeletons (e.g. peg-and-socket, ball-and-socket, etc.), T. marmorea consisting of two rigid armor plates separated by a more compliant utilizes an actuating sandwich structure consisting of two rigid ar- and actuating transverse muscle to achieve biomechanical motion. mor plates separated by a more compliant and actuating trans- The transverse muscles are protected by the ventral tegmental cal- verse muscle to achieve biomechanical motion, analogous to a lus, which probably functions to reduce the dorsal separation dis- ‘‘shear lap joint,’’ albeit one that is geometrically structured. This tance between intermediate plates. The contact of the apophyses of work also provides an understanding of how T. marmorea achieves the intermediate plates with ventral surface of their anterior the required balance between biomechanical mobility and protec- neighbors migrates laterally from the L3 cross section to the L5 tion in the passive and defensive states. In the passive state, the section as the L.C.I. of the plate assembly increases. The close prox- morphometry of the individual shell plates and plate-to-plate imity of the overlapping regions of adjacent plates appears to interconnections results in an approximately continuous curvature strongly limit lateral translation (along the ‘‘x’’-axis) and rotation and constant armor thickness and, hence, spatially homogeneous about the ‘‘y’’ axis by the intermediate plates. Longitudinal transla- protection; mobility is limited but armor coverage and protection tion (‘‘z’’ direction) of the plates is likely constrained by the compli- is maximized. When the animal is detached from a surface in the ance of the underlying muscular system and surrounding girdle. defensive state, the underlying soft tissues of the foot gain greater Although the defensive conformation (Fig. 1b) protects the under- coverage and protection by the shell plates and the animal gains lying soft parts of T. mamorea from potential predators, it creates mobility through tidal flow, but regions of vulnerability are opened vulnerability by dramatically increasing the separation distance dorsally, due to the increase in plate-to-plate separation distance of gaps 3 and 4 of the plate assembly by 75% and 100%, respec- and decrease in plate-to-plate overlap. Lastly, experiments using tively, relative to the passive, slightly curved conformation optical microscopy, scanning electron microscopy, X-ray diffrac- (Fig. 1a). Sea stars have been observed inserting their cardiac stom- tion, mercury porosimetry, fourier-transform infrared spectros- achs into the gaps between the plates of chitons that rolled into a copy, and amino acid analysis explore the microstructure and ball after being captured (Town, 1979). spatial distribution of the six layers within intermediate shell plates, the role of multilayering in resisting predatory attacks, and the detection of chitin as a major component of the intra-plate 4.6. The degree and type of armor unit overlap organic matrix and girdle. As the L.C.I. of the plate assembly increases, plate-to-plate overlap decreases and becomes more heterogeneous. In the two plate Acknowledgments assemblies with a L.C.I. of 0.43 and 0.70, the total overlap percentage of the fourth plate (relative to its long axis ‘‘l’’) ranges from We gratefully acknowledge support of the National Science approximately 60–82.5% (average = 73.9%) and 0–90% (aver- Foundation MIT Center for Materials Science and Engineering age = 44.6%), respectively, between cross sections L1 and L5. In (DMR-0819762), the US Army Research Office through the MIT Insti- addition, the average plate-to-overlap percentages (calculated tute for Soldier Nanotechnologies (Contract W911NF-07-D-0004), from projections of the plates and overlapping regions onto the the National Security Science and Engineering Faculty Fellowship ‘‘y’’–‘‘z’’ plane) of the two plate assemblies with L.C.I. of 0.43 and Program (N00244-09-1-0064), and the MIT International Science 0.70 are 62.27 ± 11.45 and 48.00 ± 9.07, respectively. The reduction and Technology Initiatives (MISTI-Israel). This work was also par- of plate-to-plate overlap, along with the aforementioned increase tially supported by the DFG (Grant No. EH 394/1-1). The authors in plate-to-plate separation distance, reduces the protection of would like to thank MIT graduate students Samuel Crawford, Chris plate assembly in the defensive conformation relative to less Ng, and Simon Choong for their assistance with photography, circle curved states. Species Cryptochiton stelleri and Chiton virgulatus fitting algorithms, and mercury porosimetry, respectively. We also have occasionally been observed exhibiting a righting behavior in thank E. Rosseeva for great technical assistance. which the plate assemblies undergo a concave curvature and slight twisting motion when the chitons were dislodged and turned up- Appendix A. Supplementary data side down by hand, or an aquarium water jet. However, Cryptochi- ton stelleri always rolled into ball when dislodged and vigorously Supplementary data associated with this article can be found, in stimulated by touch. Perhaps in some species the defensive confor- the online version, at doi:10.1016/j.jsb.2011.12.019. mation serves as a last resort in the presence of imminent threats when the chiton does not have enough time and/or security to right itself when dislodged. References

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