766 Z. Kristallogr. 2012, 227, 766–776 / DOI 10.1524/zkri.2012.1532 # by Oldenbourg Wissenschaftsverlag, Mu¨nchen
Correlation of structure, composition and local mechanical properties in the dorsal carapace of the edible crab Cancer pagurus
Helge-Otto Fabritius*,I, Eva Simone KarstenI, Keerthika BalasundaramI, Sabine HildII, Katja HuemerII and Dierk RaabeI
I Max-Planck-Institut fu¨r Eisenforschung GmbH, Department Microstructure Physics and Alloy Design, 40237 Du¨sseldorf, Germany II Johannes Kepler University Linz (JKU), Department of Polymer Science, 4040 Linz, Austria
Received February 29, 2012; accepted June 26, 2012 Published online: August 27, 2012
Crustacean cuticle / Structure-property relations / Introduction Biomineralization In recent years, biological hard tissues have received in- Abstract. The exoskeleton of crustaceans is formed by creasing attention by materials science as sources of in- the cuticle, a chitin-protein-based nano-composite with hier- spiration and templates for the development of novel mate- archical organization over at least eight levels. On the mo- rials with tailored properties that can perform new, lecular level, it consists of chitin associated with proteins unusual or multiple functions. The body of crustaceans is, forming fibres, which are organized in the form of twisted like that of all other Arthropoda, covered by a cuticle plywood. On the higher levels, the twisted plywood orga- which is produced by the outer epithelium and serves as nization is modified and forms skeletal elements with ela- an exoskeleton (Hadley, 1986). The crustacean cuticle can borate functions. The load-bearing parts of crustacean cuti- be regarded as an organic/inorganic composite based on a cle are reinforced with both crystalline and amorphous matrix formed by the structural biopolymer chitin asso- biominerals. During evolution, all parts of the exoskeleton ciated with proteins and a mineral phase that consists were optimized to fulfill different functions according to mainly of calcium carbonates and phosphates. Structurally, different ecophysiological strains faced by the animals. the cuticle is hierarchically organized over at least eight This is achieved by modifications in microstructure and levels from the molecular scale to the fully differentiated chemical composition. In order to understand the relation- structure (Fabritius et al., 2009). Molecules of the sugar ship between structure, composition, mechanical properties N-acetylglucosamine polymerize to form anti-parallel and function we structurally characterized cuticle from the chains of a-chitin. Crystallized chitin chains (Carlstro¨m, dorsal carapace of the edible crab Cancer pagurus using 1957) coated with a protein matrix form about 5–7 nm light and scanning electron microscopy (SEM). The local thick nanofibrils. The nanofibrils further assemble to chit- chemical composition was investigated using energy dis- in-protein fibres with diameters of 50–250 nm. These fi- persive X-ray spectroscopy (EDX) and confocal m-Raman bres arrange with parallel long axes to form horizontal spectroscopy. Nanoindentation tests were performed to planes. In stacks of these planes, the fibre orientation study the resulting local mechanical properties. The results changes helically and generates a twisted plywood struc- show local differences in structure on several levels of the ture (Bouligand, 1972) that forms the three main layers of structural hierarchy in combination with a very heteroge- the cuticle, the exocuticle, endocuticle and membraneous neous mineralization. The distal exocuticle is mineralized layer. Together with the external thin epicuticle, they form with calcite, followed by a layer containing a magnesium, the material that constitutes skeletal elements which can phosphate and carbonate rich phase and ACC in the prox- have different functions (Roer and Dillaman, 1984). The imal part. The endocuticle contains magnesian calcite and physical properties of cuticle are adapted to these func- ACC in special regions below the exocuticle. Structure tions. This is achieved by altering structure and composi- and mineral phase are reflected in the local stiffness and tion at different hierarchical levels utilizing the morpholo- hardness of the respective cuticle regions. The heterogene- gical and genetic prerequisites available to the organism. ity of structural organization and mechanical properties To understand the underlying design principles, it is neces- suggests remarkable consequences for the mechanical be- sary to study the relationship between structure, composi- haviour of the bulk material. tion and the resulting properties. The crustacean cuticle has been shown to possess excellent mechanical properties (Sachs et al., 2006a; Nikolov et al., 2010; 2011). The me- chanical response of the bulk material to external loads is the integral sum of the mechanical properties and behav- iour of the individual composite constituents as well as * Correspondence author (e-mail: [email protected]) their hierarchical structural arrangement on different length Cancer pagurus dorsal carapace cuticle 767 scales (Fabritius et al., 2009). In addition to the structural cuticle surface to expose the cross section and the trans- hierarchy, the mechanical properties are also influenced by verse section in different depths. For SEM, the samples the organization of the pore canal system (Sachs et al., were mounted on standard aluminium holders, rotary sha- 2008) and type and grade of mineralization (Sachs et al., dowed with a 3 to 4 nm thick platinum layer (Gatan PECS 2006b). The cuticle of crustaceans has been shown to con- 682) and viewed in a Zeiss Gemini 1540XB dual beam tain different types of minerals like magnesium calcite, microscope at an acceleration voltage of 5 kV using a amorphous calcium carbonate (ACC) and also amorphous small aperture (30 mm) and an in-lens detector. Where ne- calcium phosphate (ACP). They can occur together in the cessary, samples were etched superficially using aqueous same layers but also be restricted to specific regions of the EDTA solution (0.15M, 15 min) followed by a quick wash cuticle (Levi-Kalisman et al., 2002; Al-Sawalmih et al., in H2O bidest and 100% methanol for 1 s each. To obtain 2008; Hild et al., 2008). A number of recent studies have images at higher magnification, a set of samples was criti- correlated the anisotropy in structure, mineral content and cal point dried (Baltec CPD 030), rotary shadowed with composition of cuticle from different crustacean species 2 nm of platinum (Balzers BAF 300) and viewed in a Hi- with evolutionary adaptations to different habitats and de- tachi S-5200 FESEM at an acceleration voltage of 2 kV. fence strategies that impose different mechanical require- For LOM, EDX and m-Raman spectroscopy, air dried ments (Boßelmann et al., 2007; Neues et al., 2007; Hild samples were glued to standard aluminium stubs or, for et al., 2009). However, in contrast to other mineralized nanoindentation, to round magnetic steel discs with the biological materials like bone (Gupta et al., 2006) little is surface of interest oriented parallel to the support. Subse- known about the influence of the local structure and quently, these surfaces were polished to the desired depth mineralization differences on mechanical properties like using an ultramicrotome (Power-Tome PT-XL, Labtec) stiffness and hardness. This is especially true for the lower equipped with a diamond knife (Diatome). The feed rate hierarchical levels like the different layers constituting the was successively reduced from 1 mm to 10 nm to obtain a cuticle. A previous study using nanoindentation on air- low surface roughness. Light optical micrographs were re- dried cuticle of the lobster Homarus americanus has corded in bright field mode on a Leica DM 400M micro- shown that the elastic properties are mainly affected by scope. the grade of mineralization (Sachs et al., 2006b). In terms of structural organization, the cuticle forming Energy-dispersive X-ray spectroscopy the dorsal carapace of the large decapod crustacean Cancer pagurus, the edible crab, has been shown to be remark- For EDX analysis, polished samples were coated with 3 nm ably complex by a number of early investigations (Drach, of platinum to ensure conductivity. Elemental maps were 1939; Hegdahl et al., 1975a–c). In an attempt to analyse recorded using the EDAX system (PV7716/08 ME) at- the relationships of structure, mineralization and mechani- tached to the SEM (Zeiss Gemini 1540XB). The post-pro- cal properties in the individual layers of the dorsal cara- cessing of the maps including background correction was pace of C. pagurus, we combined light optical microscopy performed using the Genesis software package (EDAX). (LOM), scanning electron microscopy (SEM), energy- dispersive X-ray spectroscopy (EDX), scanning confocal Scanning confocal m-Raman spectroscopy m-Raman spectroscopic imaging (SCm-RSI) and nanoin- dentation testing. The results show that the two mechani- Raman spectral images were recorded on microtome po- cally relevant layers exo- and endocuticle differ both in lished cross sections of carapace cuticle with a confocal the structural organization and the type of mineralization, Raman microscope (WITec Alpha300 R) using a Nd-YAG which is reflected in the mechanical properties. Addition- laser (wavelength 532 nm, 17 mW) and a Nikon objective ally, the complex structure and local gradients in composi- lens (100 ,Na¼ 0,925). In the areas of interest, Raman tion of the cuticle indicate strong implications on the frac- spectra between 0 and 3750 cm 1 were recorded line- ture behaviour, especially on the distribution of imposed by-line with a raster of 25 pixels per 100 mm2 at an inte- loads and the propagation of cracks. gration time of 100 ms. For spectral analysis the peaks were indexed according to earlier measurements on stand- ard samples by Hild et al. (2008). The Raman spectral maps Experimental were obtained by integrating over specific bands after background correction and plotting the values in x–y coor- Sample preparation dinates using the WITecProject 2.02 software.
Large, adult specimens of the European edible crab Can- Nanoindentation cer pagurus in intermolt stage were obtained from a local seafood supplier. All samples were prepared from the dor- Nanomechanical characterization was performed with a sal carapace, which was dissected immediately and stored Hysitron Tribo-Indenter (Hysitron Inc.) that was operated frozen at 20 C to prevent natural decay processes. For in a closed loop feedback system, which controls the load investigation of the microstructure, small rectangular applied to the indenter by means of an electrostatic trans- pieces were cut from the carapace using a jeweller’s saw. ducer. A spherical indenter with a 5 mm tip radius was The specimens were air-dried and either fractured or po- used. The area function describing the indenter profile was lished to the desired depth using an ultra-microtome. Sam- determined in precise calibration measurements using a ples were prepared both perpendicular and parallel to the polymethyl-methacrylate (PMMA) standard sample. All 768 H.-O. Fabritius, E. S. Karsten, K. Balasundaram et al. indents were performed using displacement control mode, to a depth of 300 nm using a trapezoidal loading function. The indentation depth was increased to the maximum depth in a time interval of 5 s, dwelled at maximum depth for 5 s and unloaded in 5 s. The indents were distributed at regular distances in rectangular patterns with dimen- sions corresponding to the sizes of the chosen areas of interest. All experiments were performed at room tempera- ture. The local reduced modulus (Er) and hardness (H)of the material were determined using the analysis method of Oliver and Pharr (1992).
Results
Microstructure of the dorsal carapace The cuticle of the dorsal carapace of C. pagurus com- prises four distinct layers: from distal to proximal the epi- cuticle, exocuticle, endocuticle and a membraneous layer. Depending on the size of the animal and the probed loca- tion its thickness varies between 1.2 and 2 mm. Light mi- crographs of polished sections show striation patterns ori- ginating from the twisted plywood structure that allow to study the spatial progression of the stacked fibres in the different layers (Fig. 1). The epicuticle at the distal margin of the cross section (Fig. 1A) is visible as a thin continu- Fig. 1. Structural organization of cuticle from the dorsal carapace of ous dark line. The subjacent exocuticle has a dark, or- C. pagurus on the bulk level. (A–C) Light micrographs of microtome ange-reddish pigmentation and makes up for about 25% of polished cross section (A) and transverse sections through the exo- the total cuticle thickness. Its distal parts appear more or (B) and endocuticle (C), showing the progression of the fibre layers less unstructured, while the proximal areas show a fine, forming the exocuticle (exo), endocuticle (endo) and membraneous narrow horizontal striation (Fig. 1A, B). The exocuticle is layer (ml). epi: epicuticle; eb: endocuticular bulge. interrupted in irregular distances by parts of the endocuti- cle that bulge out in distal direction. This results in a layer penetrated by circular holes of varying diameters (Fig. 1A, a blocky appearance with sharp, rectangular borders. B). A distinct thin and dark line marks the transition be- Superimposed layers rotate at large angles. This results in tween exo- and endocuticle. The major part of the endocu- the completion of a 180 rotation in a stacking height of ticle is horizontally striated with the stacking height of the about 1.3 mm. Mineral particles of different sizes and irre- plywood layers gradually decreasing from distal to proxi- gular shapes are distributed between mineralized fibrils. mal. The parts located under the exocuticle show a narrow, They occur frequently in areas where the fracture mode of wavy striation while those under the bulges are evenly the fibrils indicates the presence of a pore canal. Indivi- striated. In the bulge areas themselves, the plywood layers dual, clearly distinguishable pore canals are not observed gently curve towards the outer surface of the integument (Fig. 2A). In the proximal part of the exocuticle the stack- where they proceed vertically. The centre of the bulges is ing height increases. The mineralized fibrils appear formed by a zone shaped like an upside down cone which smoother and their fractured edges are less well defined does not show any striation (Fig. 1A). On samples po- than in the distal area. Pore canals can now clearly be lished transversely through the exocuticle, the bulges are distinguished. They have a nearly circular cross section, visible as circular structures encompassed by irregularly diameters of around 300 nm and seem to be devoid of or- striped areas (Fig. 1B). Circular, sometimes even spiralling ganic tubes and mineral particles (Fig. 2B). Close to the patterns are observed on transversely polished surfaces of transition to the endocuticle, the exocuticle becomes less lower areas within the endocuticle (Fig. 1C). Proximally, dense. The fibrils form smooth, band-like bundles that the cuticle is bordered by the unstructured membraneous show no obvious signs of mineralization. In addition to layer (Fig. 1A). the vertical pore canals, the structure is pervaded by large, Ultrastructurally, the epicuticle consists of an about horizontal channels with apparently random orientation. 1 mm thick solid outer layer and a 3–4 mm thick inner The transition between exo- and endocuticle is abrupt layer. The inner layer contains numerous small pore canals (Fig. 2C). The entire endocuticle consists of twisted ply- (Fig. 2A) oriented perpendicular to the cuticle surface. wood composed of hollow mineral tubes containing organ- Sometimes, these pore canals contain small mineral parti- ic fibres. The tubes are fused and form a compact struc- cles. The inner epicuticle is followed by the first layers of ture interrupted only by pore canals. Depending on the the exocuticle. In its distal part, individual plywood layers orientation of the tubes, they fracture either perpendicular can be distinguished. The fractured mineralized fibrils have or split along their long axes (Fig. 2C–E). Cancer pagurus dorsal carapace cuticle 769
Fig. 3. (A) Microstructure of the endocuticle showing clearly defined chitin-protein fibres (cf) composed of nanofibrils (nf) and surrounded by mineral particles creating tubular structures (mt), which are fused with neighbouring tubes. (B) Transversely fractured endocuticle show- ing mineralized fibres undulating around pore canals with biconvex cross sections. (C) Pore canal (pc) with reinforcing mineralized fibres (white lines) located in the narrow edges which reduce the volume and leave an oval cross section.
Fig. 2. Microstructure of the different cuticle layers. (A) Cross frac- (Fig. 3B). The opposite ends of the long axis are filled ture through the junction between the outer (epi-1) and inner epicuti- with mineralized chitin-protein fibres that resemble the cle (epi-2) and the strongly mineralized distal layers of the exocuticle horizontal fibres forming the plywood (Fig. 3C). They (exo). (B) Mineralized fibres in the central region of the exocuticle with narrow pore canals (arrows). (C) Transition between the loosely flank the pore canals along their entire length within the structured proximal part of the exocuticle and the endocuticle (endo). endocuticle, leaving an open lumen with an oval cross sec- (D) Twisted plywood arrangement of chitin-protein fibres surrounded tion. The pore canals contain long flexible tubes consist- by fused mineral tubes typical for endocuticle. The fractured pore ing of organic material (Figs. 2C–E, 3B) that shrink upon canals expose tubes consisting of organic fibres shaped like twisted drying of the samples. The long axes of the pore canals ribbons (arrows). (E) Transition between endocuticle and membra- neous layer (ml). Arrows: pore canals. rotate together with the horizontal fibre layers, therefore the tubes frequently look like twisted ribbons (Fig. 2D). The membraneous layer consists of stacked layers of un- High resolution electron micrographs reveal that the or- mineralized fibres with a smooth appearance (Fig. 2E). ganic matrix in the endocuticle consists of clearly defined At the margins of the bulges, the otherwise horizontal chitin-protein fibres with an average diameter of 70 nm. plywood layers of the exocuticle become thinner and The fibres are composed of 7 nm thick nanofibrils curve upwards to proceed vertically parallel to the bulge (Fig. 3A). Every fibre is surrounded by mineral granules (Figs. 1A, 4A). At the top, the stacking height further de- with sizes ranging from 20 to 50 nm creating a tubular creases. This thin layer can be observed to cover the entire structure. Neighbouring tubes are densely packed and the bulge on gently decalcified samples (Fig. 4A). In untreated interstitial space is filled with mineral (Fig. 3A). Transverse samples, this could not be distinguished due to the fact fractures through the endocuticle show that the mineral- that it was not possible to obtain a fracture through the ized fibres undulate around pore canals. These canals have centre of a bulge. The pore canals are continuous over the an elongated, biconvex cross section with dimensions of transition between endo- and exocuticle and maintain their about 2 mm in the long axis and 500 nm in the short axis orientation perpendicular to the plywood layers, but follow 770 H.-O. Fabritius, E. S. Karsten, K. Balasundaram et al.
Fig. 5. Electron probe microanalysis (EDX) of cross fractured and microtome polished cuticle from the dorsal carapace of C. pagurus. (A) SEM micrograph and qualitative X-ray spectrum showing the presence of C, O, Mg, P, and Ca. (B–D) Spectral maps showing the distribution of calcium (B), magnesium (C) and phosphorus (D).
Elemental distribution EDX spectra recorded on polished cross sections through the dorsal carapace show the presence of carbon, oxygen calcium, magnesium and phosphorous (Fig. 5A). Elemental Fig. 4. Microstructure of the endocuticle in different areas of the mapping shows uniformly high calcium signals throughout bulge regions (see light micrograph). (A) Fracture through the exo- the endocuticle including the bulge areas. The high calcium endocuticle junction in a bulge close to the cuticle surface showing (Fig. 5B) signal extends laterally into the most distal layer the thin exocuticle (exo) covering the bulge centre (endo bulge), the of the exocuticle. Proximally, calcium signal intensity gra- sample was gently etched using 0.15 M EDTA solution. (B) Structure of the heavily mineralized fibres in the centre of a bulge. (C) Transi- dually decreases. No calcium was detected in a narrow hor- tion between exo- and endocuticle at the base of a bulge. The layers izontal band located at about three quarters of the total delaminated due to the drying process. The pore canals (white ar- thickness of the exocuticle. The most proximal layer of the rows) can be seen to traverse the junction continuously (insert). (D) exocuticle shows a uniform calcium signal which is slightly In bulge areas the mineralized fibres in the plywood layers rotate vertically (indicated by black arrows), but the pore canals (white ar- lower than in the endocuticle. Magnesium (Fig. 5C) is rows) maintain their orientation perpendicular to the cuticle surface. evenly distributed in the endocuticle including the bulges, except for the areas situated under the exocuticle where the the changes of their course (Fig. 4C). In the endocuticular signal is significantly lower. These areas extend over half plywood layers that form the bulges, the mineralized fi- of the total endocuticle thickness in proximal direction. The bres share the same structure than the rest of the endocuti- highest signals are recorded in the distal parts of the exo- cle. Due to the change in course, the fibre layers rotate cuticle. The proximal quarter of the exocuticle is devoid around a horizontal axis. In these areas, the pore canals do of magnesium. Phosphorous (Fig. 5D) is restricted to the not change direction and pervade the vertical plywood par- distal parts of the exocuticle, excluding a thin layer adja- allel to the in-plane direction of the fibre layers (Fig. 4D). cent to the epicuticle where no signal is present. In the centres of the bulges the fibres appear to be heavily mineralized. Therefore, regular plywood as in the lower Raman imaging regions is indistinguishable and individual fibres can only occasionally be observed. Here, the pore canals are small, The Raman spectral map of the distribution of total carbo- with circular cross sections and frequently filled with nate (Fig. 6A, A*) was obtained by integrating over the mineral (Fig. 4B). spectral area ranging from 1050 to 1125 cm 1 which is Cancer pagurus dorsal carapace cuticle 771
ting the intensity of the integral values of the phosphate band (Sauer et al., 1994) between 935 and 985 cm 1 (Fig. 6F, 3). Phosphate is prevalent in the exocuticle, ex- cept for the most distal part where the signal faints and disappears. Low phosphate signals are present in the rest of the cuticle. The lowest intensities are observed in the endocuticle areas located under the exocuticle (Fig. 6C). The most characteristic peaks described for the organic matrix are situated in a band ranging from 2800 to 3025 cm 1 (Fig. 6F, 4). The resulting maps (Fig. 6D, D*) show high signals in a thin horizontal line representing the mostly unmineralized epicuticle. The distal part of the exocuticle shows no signal for organic material. Proxi- mally, the signal increases to fade again in the lower quar- ter of the exocuticle. At the transition between exo- and endocuticle we observed a thin line of elevated signal. The upper half of the endocuticle shows slightly lower signals than the lower half including the bulge area. In both parts, the intensities are uniform. Carotene distribution maps were obtained by plotting the intensity of the integral val- ues of two characteristic bands: The first band located be- tween 1140 and 1235 cm 1 includes the C––C in plane single bond stretching mode. The second band between 1450 and 1580 cm 1 includes the C¼C stretching mode and is shown in Fig. 6E, E* (Saito et al., 1983). The spec- tral images show that carotenes are present in the whole cuticle. The highest signals are observed within the distal quarter of the exocuticle, with slightly lower intensities in the proximal parts. The endocuticle area located under the exocuticle shows a clear signal and small amounts also appear within the other regions.
Nanoindentation tests Variations of the local mechanical properties were investi- gated by distributing rectangular arrays of nanoindents on Fig. 6. Raman-spectroscopic maps of the full cross section (A–E) and a bulge area (A*–E*) recorded on cross fractured and microtome cross- and transverse sections of cuticle. This allowed polished cuticle from the dorsal carapace of C. pagurus. The maps creating surface plots of the reduced elastic modulus (Ered) show the local distribution of carbonate (A, A*), calcite (B, B*), (Fig. 7) and the hardness (H) (Fig. 8) of selected areas. phosphate (C), organic material (D, D*) and carotene (E, E*). (F) Every rectangle in the surface plots represents the area Cumulated Raman spectra recorded in six different locations marked around an individual indent located in the centre. The col- by Roman numerals I to VI in (B). The spectra show differences in the peaks typical for carbonate (1, orange), calcite (2, red), phosphate our coding corresponds to the obtained values for Ered and (3, blue), organic material (4, green), and carotene (5, purple). H. The results show that both stiffness and hardness fol- low the same trends in the probed areas. On the cross sec- tion of the cuticle, the indent patterns cover its full thick- 2 characteristic for [CO3] stretching vibration (Fig. 6F, 1). ness and were set alternative in areas where bulges and Carbonate signals with high intensity are present all over exocuticle are exposed (Figs. 7A, 8A). For each pattern, the endocuticle and especially in the centres of the bulges. the Ered and H values from indents in the same horizontal In the exocuticle, elevated carbonate signals are observed row were averaged and plotted as a function of their verti- in a distal layer comprising 25% of its total thickness. cal position on the sample starting at the proximal end Proximally, their intensity decreases gradually. The distri- (Figs. 7B, 8B). The curves show that the properties are bution of calcite was obtained by plotting the integral val- relatively uniform throughout the lower half of the endo- ues of the spectral region from 268 to 310 cm 1 which cuticle with an average stiffness of about 10 GPa and includes the calcite lattice vibration at 280 cm 1 (Fig. 6F, hardness of about 200 MPa. Further distal, the areas under 2). The obtained maps (Fig. 6B, B*) show that the entire bulges are slightly stiffer and harder than those under the endocuticle contains calcite except for the areas situated exocuticle. In the bulges themselves, both values increase under the exocuticle. These areas extend over half of the dramatically to a maximum of 40 GPa in stiffness and total endocuticle thickness. Calcite is also present in the 1200 MPa in hardness, followed by a small drop at the outermost layer of the exocuticle. Proximally, the signal distal margin of the cuticle. In the areas under the exocuti- fades into line-like features aligned perpendicular to the cle, stiffness and hardness gradually decrease and reach cuticle surface. The phosphate map was obtained by plot- the lowest values in the central regions of the exocuticle, 772 H.-O. Fabritius, E. S. Karsten, K. Balasundaram et al.
Fig. 7. Local variation of the reduced elastic modulus Ered obtained Fig. 8. Local variation of the hardness H obtained by nanoindenta- by nanoindentation plotted on the tested knife polished cross section tion plotted on the tested knife polished cross section (A) and trans- (A) and transverse section through the exocuticle (C). The colour of verse section through the exocuticle (C). The colour of every rectan- every rectangle in the surface plots represents the value calculated gle in the surface plots represents the value calculated from the indent from the indent located in its centre. The white circles indicate the located in its centre. The white circles indicate the starting point of starting point of an indent pattern and the arrows indicate the direc- an indent pattern and the arrows indicate the direction of the indivi- tion of the individual indent rows. (B) The curves represent the aver- dual indent rows. (B) The curves represent the averaged H of indents aged Ered of indents in the same horizontal row of patterns I, II and III in the same horizontal row of patterns I, II and III in (A) plotted as a in (A) plotted as a function of distance from the proximal margin of function of distance from the proximal margin of the cuticle to illus- the cuticle to illustrate the trends over the structurally differing areas. trate the trends over the structurally differing areas. with about 5 GPa for Ered (Fig. 7B) and 100 MPa for H (Fig. 8B), respectively. In the endocuticle, the maps show Discussion bands of elevated stiffness and hardness that correspond well to the progression of the underlying plywood layers. One of the most notable features of the cuticle forming Directly below the exocuticle, the values are more uni- the dorsal carapace of the crab C. pagurus is the fact that form. In the exocuticle, the values are uniformly low ex- the exocuticle is interrupted by parts of the endocuticle cept for the most distal areas which show narrow horizon- that bulge out in distal direction (Fig. 1A, B) (Hegdahl tal bands of elevated stiffness and hardness. In the bulges, et al., 1977a). In many other crustacean species, the exo- it becomes obvious that the very high values for stiffness cuticle is a continuous layer composed of horizontally ar- and hardness are confined to the central zone and corre- ranged fibre layers (Raabe et al., 2006; Hild et al., 2008). spond to its shape which resembles an upside down cone The progression of the plywood layers in the dorsal cara- (Figs. 7A, 8A). The samples which were indented perpen- pace of C. pagurus shows that the bulges consist of co- dicular to the surface were polished down to a depth of nically raised endocuticular layers. This results in concen- 200 mm, exposing the circular bulge areas surrounded by trically arranged plywood reminding of the structure of exocuticle. Patterns of indents were placed both on bulges osteons in the bones of vertebrates (Weiner and Wagner, and in exocuticle areas. The results correspond well with 1998). Circular, sometimes even spiralling patterns ob- the corresponding region on the cross sections. The high- served on transversely polished surfaces of lower areas in est values for stiffness and hardness are observed in the the endocuticle show that the bulges can be traced even centres of bulges and gradually decrease towards their into the areas formed last (Fig. 1C). margins. In the exocuticle, the values are much lower and The thin epicuticle that covers the whole integument fluctuate concurrent with structural features on the sam- contains no helicoidally arranged fibres and consists of a ples (Figs. 7C, 8C). compact layer and a subjacent layer with numerous pore Cancer pagurus dorsal carapace cuticle 773 canals as described earlier (Hegdahl et al., 1977c) (Fig. 2A). material. Within this region the Raman image of calcite Our Raman spectroscopic results show that the compact appears bright (Fig. 6B). The corresponding average spec- outer layer consists mainly of epicuticular waxes, as indi- trum (Fig. 6F/II) shows peaks at 280 and 1088 cm 1 con- cated by a band in the range from 2800 to 3000 cm 1 that firming the presence of calcite. This mineral phase extends appears in the averaged spectra of this region (Fig. 6D, F/I). towards proximal forming line-like features aligned per- This position is characteristic for C––H stretching bands pendicular to the surface. This is consistent with earlier appearing in hydrocarbons. The absence of bands at high- findings of Hegdahl (1977b), who described the outermost er wave numbers that are characteristic for proteins or layers of exocuticle as heavily mineralized. The line-like chitin supports this assumption. The pore canals constitut- extensions of the calcitic region correspond to the inter- ing the inner layer are found to contain mineral particles prismatic areas described by earlier investigators (Drach, (Fig. 2A), which are visible as a very fine horizontal line 1939; Hegdahl et al., 1975a–c) and originate from the im- in the respective Raman spectroscopic image (Fig. 6A, prints left by epithelial cells during formation of the cuti- carbonate). This line is absent on the image obtained by cle tissue. In the central region below, the EDX measure- integration over the band containing the calcite lattice vi- ments (Fig. 5C, D) show high contents of magnesium and bration peak at 280 cm 1 (Fig. 6B, F/I), indicating that the phosphorus, but little calcium. Raman imaging reveals that mineral particles are not calcite. Ultrastructural investiga- both carbonate and phosphate are present at the same time tion of the soft membraneous layer which covers the prox- (Fig. 6A, C). The position of the carbonate peak in the imal surface of the cuticle shows that it consists of regu- averaged spectrum (Fig. 6F/III) is shifted to the lower larly layered fibrous planes. They are composed of chitin value 1083 cm 1. Since the peak at 280 cm 1 is absent, it and proteins (Welinder, 1975) and show no sign of miner- can be concluded that amorphous calcium carbonate alization. The strong calcium signal visible in the EDX (ACC) is present in this area. The small peak at 956 cm 1 mapping (Fig. 5C) was identified to be an artefact. During suggests that in this area octa-calcium phosphate appears. microtome polishing of the samples, the diamond knife The high magnesium and comparably low calcium values pushed debris from the cuticle sections into the loose shown by elemental mapping (Fig. 5B, C) also suggest the structure of the membraneous layer. Both epicuticle and formation of an Mg––PO4––CO3 phase in this region. membraneous layer were not included into mechanical Concurrently, a strong band in the range from 2800 to testing. The epicuticle was left out due to its small thick- 3000 cm 1 appears (Fig. 6F/III) which can give evidence ness and irregular progression. In case of the membra- for the presence of organophosphates (Welinder, 1975). In neous layer, it was not possible to obtain a polished sam- the most proximal region of the exocuticle, EDX measure- ple where it was not damaged or partially removed during ments show only low magnesium and phosphorus signals, preparation. Since both layers represent only a tiny frac- but high calcium signals. The Raman spectral images indi- tion of the bulk of the cuticle, it seems unlikely that they cate the presence of carbonate (Fig. 6A) and, to a lower play a significant role in the overall mechanical properties extent also phosphates (Fig. 6B). The content in organic of the dorsal carapace. material is reduced (Fig. 6D). Determine the positions of From an in-plane point of view of the full dorsal cara- the phosphate peak reveals a shift to the higher value pace cuticle, the exocuticle forms two fundamentally dif- 959 cm 1, indicating the presence of hydroxyapatite as fering types of macroscopic structure: The regions where phosphate-containing mineral in this region (Fig. 6F/IV). it is present as a layer comprising one fourth of the whole The absence of calcite signals (Fig. 6B) and the peak at cuticle thickness (Fig. 1A, B) and those where it covers 280 cm 1 (Fig. 6F/IV) indicate that ACC is the main car- the endocuticular bulge structures (Fig. 4A). Interestingly, bonate mineral here. The microstructure of both regions the fibre layers constituting the exocuticle are nowhere shows a rather smooth appearance with little evidence for interrupted. The transformation between the two regions larger mineral particles. This suggests that mineral parti- happens by changes in direction of the plywood layers cles are nanoscopic and interspersed between individual bordering the bulges and a significant reduction in stacking chitin nanofibrils (Boßelmann et al., 2007). A similar or- height where the exocuticle covers the bulges (Fig. 4A, C). ganization of the mineral phase has been found in both Thus, the marginal fibre layers of the exocuticle proceed exo- and endocuticle of other large decapod crustaceans parallel to those of the endocuticle forming the bulges. (Raabe et al., 2006; Fabritius et al., 2009). The Raman This explains why the pore canals are continuous between imaging shows that in addition to chitin and proteins, caro- both layers (Fig. 4C). From distal to proximal, the exocu- tenes are present in the whole exocuticle (Fig. 6E, F) The ticle can be subdivided into three regions differing both in highest signals are observed within the distal part of the microstructure and mineral composition. In the most distal exocuticle, which is consistent with the coloured appear- region subjacent to the epicuticle, including the parts ance of this region in light micrographs (Fig. 1B). above bulges, the plywood structure is heavily mineralized Nanoindentation experiments on corresponding areas of with spherical and elongated particles of different sizes. the exocuticle’s cross section show that these differences The particles also fill up most of the pore canals (Fig. 2A). in mineralization and structure are directly reflected in the Individual organic fibres can not be distinguished. The local stiffness (Fig. 7A, B) and hardness (Fig. 8A, B) of EDX measurements show that this region contains both the tissue (Fig. 5A). The outermost calcitic layer of the calcium and magnesium, but very little phosphorus. In the exocuticle shows the highest values which correspond well Raman map of organic material obtained by integrating to values obtained from the exocuticle of the lobster over the spectral area from 2850 to 3000 cm 1 this region Homarus americanus. Lobster exocuticle has a similar mi- appears dark, indicating a very low content of organic crostructure and is mineralized with nanoscopic calcite 774 H.-O. Fabritius, E. S. Karsten, K. Balasundaram et al. particles (Sachs et al., 2006b). In the phosphate-rich cen- The nanoindentation maps on the cross section of the tral regions the values are lowest and consistent with com- endocuticle show that both local stiffness (Fig. 7A, B) and parable artificial materials like calcium phosphate/polymer hardness (Fig. 8A, B) are very uniform throughout the en- composites (Kikuchi et al., 1997). In the ACC-rich proxi- docuticle with the exception of the bulge centres. This is mal region, the average Ered of about 5 GPa and hardness surprising, since calcite has been shown to have signifi- of about 100 MPa are slightly lower than in the similarly cantly better mechanical properties in crustacean cuticle structured endocuticle of H. americanus (Sachs et al., than ACC (Sachs et al., 2006b). The uniformity of stiff- 2006b). This is probably due to the loose structure and the ness and hardness in areas mineralized with calcite and abundance of large horizontal channels within this region ACC, respectively, suggests that the mechanical properties (Figs. 2C, 4C). are not determined by the mineral phase, but rather by On the fibre level, exo-and endocuticle differ in the differences in structural organization. Since the structural organization of matrix and mineral. The organic fibres in organization on the fibre level is similar this must be the exocuticle are always associated with mineral and not caused by modifications on higher levels of hierarchy. One well defined (Fig. 2A–C). The endocuticle consists of possibility is the lower stacking height and irregular pro- clearly defined chitin-protein fibres composed of indivi- gression of the twisted plywood layers observed in the dual nanofibrils (Fig. 3A), which are devoid of mineral ACC-rich areas (Fig. 1A), which would increase the influ- particles. Every fibre is surrounded by small mineral ence of the organic fibres on the measured modulus and particles creating a tubular structure which fuses with hardness, since their long axis is much stiffer than the neighbouring tubes to create a solid block of material sur- transverse axis (Nikolov et al., 2010; Fabritius et al., 2011). rounding the pore canals (Fig. 2D, E) (Hegdahl et al., Since we used a rather large spherical indenter (5 mm), 1977a). A similar fibre organization occurs in a number indentations in this area would cover more fibres oriented of different Brachyura (true crabs) species (Giraud-Guille, with their long axis parallel to the loading direction. Var- 1984; Dillaman et al., 2005), but has not been described iations in Ered and H in the calcite-rich areas of the endo- for other Decapoda (Raabe et al., 2006) or other major cuticle are observed to correspond quite well to the pro- crustacean taxa like Isopoda (Hild et al., 2008, 2009; gression of the plywood layers. This further supports an Seidl et al., 2011). This type of microstructure is found influence of the plywood organization (Figs. 7A, 8A). The throughout the endocuticle including the bulges, where centres of the bulges are extremely stiff and hard. This can the helicoidally organized mineralized fibre layers change be observed concurrently on cross and transverse sections their orientation from horizontal (Fig. 2D) to vertical of the cuticle (Figs. 7A, C; 8A, C). It is caused by the high (Fig. 4D). Interestingly, the organization of the pore canal grade of mineralization together with low organic material system is not affected by this modification. The pore ca- content and the almost complete absence of open pore ca- nals resemble those of H. americanus in shape and size nals which makes these regions structurally very compact. (Raabe et al., 2006; Sachs et al., 2008). However, their This is in accordance to earlier works that show that the effective volume is greatly reduced by mineralized fibres grade of mineralization has a strong influence on the result- located in the narrow edges which reinforce the canals ing mechanical properties of cuticle (Sachs et al., 2006b). over their entire length leaving an oval cross section Both structural organization and type and degree of (Fig. 3B, C). mineralization observed in cuticle from different Crustacea EDX and Raman spectroscopic analysis reveal that, de- species have been positively correlated to ecophysiological spite the uniform microstructural organization, the minera- adaptations like defensive strategies (Becker et al., 2005; lization of the endocuticle differs in the regions located Hild et al., 2008), special habitats (Hild et al., 2008; Seidl under the exo-endocuticle junctions and the rest. Adjacent et al., 2011) or economy of material and thus energy (Fab- to the exocuticle, the Raman spectroscopic maps show the ritius et al., 2011). The organization of the cuticle forming 2 [CO3] breathing mode but no calcite lattice vibration the dorsal carapace of the crab C. pagurus suggests a high signals (Fig. 6B, F/V), this indicates the presence of ACC. resistance against mechanical loads, especially loads ap- In the lower parts, the organic chitin protein matrix is plied in the direction perpendicular to the surface. This mineralized with calcite, as indicated by the Raman spec- can be interpreted as a protective adaptation against their troscopic results for carbonate and calcite (Fig. 6A, B, F/VI). main predators, various species of octopus (Mollusca, Ce- This is also the case in the bulge regions (Fig. 6A*,B*), phalopoda). The alternation of the extremely hard bulges but the signals for organic material are much lower and the calcitic distal layers of exocuticle on the dorsal (Fig. 6D*). This is consistent with the very few organic surface of the carapace and all other exposed areas of the fibres observed within the mineral phase of bulge regions exoskeleton of C. pagurus provide an effective barrier (Fig. 4B). The elevated magnesium signal shown by ele- against penetration of sharp and pointy objects like the mental mapping (Fig. 5C) is consistent with an earlier che- beaks of cephalopods (Miserez et al., 2007). Furthermore, mical analysis of the entire carapace cuticle (Boßelmann the bulges can act like pillar structures which transfer com- et al., 2007) that demonstrated the presence of magnesian pressive loads to lower regions of the endocuticle. There, calcite in C. pagurus. The Raman imaging shows that the transition from vertical to horizontal arrangement of carotenes are present in the whole endocuticle (Fig. 6E) the plywood layers can dissipate the energy laterally. The except for the central regions of bulges which are devoid marginal reinforcement of the endocuticular pore canals of carotene signals (Fig. 6E*). Clear signals are observed with mineralized chitin-protein fibres is suitable to en- mainly within the ACC region, but small amounts also hance both the resistance against compressive loads in appear within the calcite region. normal direction and in transverse direction by preventing Cancer pagurus dorsal carapace cuticle 775 the pore canals to collapse. Thus, it is very unlikely to References observe a honeycomb-like behaviour under lateral compres- A. Al-Sawalmih, C. Li, S. Siegel, H. Fabritius, S. B. Yi, D. Raabe, sion as has been described in the lobster H. americanus P. Fratzl, O. 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