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Structure, composition and properties of naturally occurring non-calcified crustacean cuticle
Arthropod Structure & Development 38 (2009) 173–178
Contents lists available at ScienceDirect
Arthropod Structure & Development journal homepage: www.elsevier.com/locate/asd
Structure, composition and properties of naturally occurring non-calcified crustacean cuticle B.W. Cribb a, b, *, A. Rathmell a, b, R. Charters a, b, R. Rasch a, H. Huang c, I.R. Tibbetts d a
The University of Queensland, Centre for Microscopy & Microanalysis, Brisbane 4072, Australia The University of Queensland, School of Integrative Biology, Brisbane 4072, Australia c The University of Queensland, School of Engineering, Brisbane 4072, Australia d The University of Queensland, Centre for Marine Studies, Brisbane 4072, Australia b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 September 2008 Accepted 13 November 2008
Crustaceans are known for their hard, calcified exoskeleton; however some regions appear different in colour and opacity. These include leg and cheliped tips in the grapsid crab, Metopograpsus frontalis. The chelipeds and leg tips contain only trace levels of calcium but a significant mass of the halogens, chlorine (Cl) and bromine (Br). In contrast, the carapace is heavily calcified and contains only a trace mass of Cl and no Br. In transverse section across the non-calcified tip regions of cheliped and leg the mass percent of halogens varies with position. As such, the exoskeleton of M. frontalis provides a useful model to examine a possible correlation of halogen concentration with the physical properties of hardness (H) and reduced elastic modulus (Er), within a chitin-based matrix. Previously published work suggests a correlation exists between Cl concentration and hardness in similar tissues that contain a metal (e.g. zinc). However, in M. frontalis H and Er did not vary significantly across cheliped or leg tip despite differences in halogen concentration. The non-calcified regions of M. frontalis are less hard and less stiff than the carapace but equivalent to values found for insect cuticle lacking metals. Cheliped tips showed a complex morphological layering that differed from leg tips. Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Chitin Polysaccharides Stiffness Nanoindentation Arthropod Crab
1. Introduction Crustaceans are well known for their hard, calcified exoskeleton, but some regions show flexibility, indicating a different structure and composition. Surprisingly little is known about these alternative cuticles. A morphological study has been carried out on intersegmental membranes, branchial and inner branchiostegal cuticles of the crab, Carcinus maenas (Compe`re and Goffinet, 1987). But these are not the only regions that appear to be non-calcified in crustaceans. The leg tips and grasping claws (cheliped tips) of grapsid crabs are of a different colour and opacity from the carapace and are rubbery to touch; characters potentially indicative of a lack of typical mineralization. Hints about composition are available from unpublished findings reported by Schofield et al. (2003) but nothing has been formally published on structure, composition, hardness or stiffness of these materials. Schofield et al. (2003) indicate that as calcium
* Corresponding author. The University of Queensland, Centre for Microscopy & Microanalysis, St. Lucia Campus, Brisbane 4072, Australia. Tel.: þ61 7 33657086; fax: þ61 7 3346 3993. E-mail address: [email protected] (B.W. Cribb). 1467-8039/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.asd.2008.11.002
levels drop, the predominant inorganic element in the tips of tarsal claws in crabs becomes chlorine (Cl). The grapsid, or grasping crab, Metopograpsus frontalis uses the tips of the chelipeds to scrape and tug algae from intertidal substrate surfaces which it then eats (Shaw and Tibbetts, 2004). These chelipeds and legs are tipped with a translucent light brown coloured material that is readily distinguished from the rest of the carapace. The current study examines the structure, composition and the physical properties of hardness and elastic modulus for this unusual material. 2. Materials and methods 2.1. Animals and behavioural observations Two species of crab, M. frontalis (Grapsidae) and Thalamita sima (Portunidae), were collected from sites around Moreton Bay, Queensland, Australia (Fig. 1A, B). While M. frontalis is a grapsid crab and shows modification of the tips of the chelipeds and legs (Fig. 1A), T. sima is a portunid or swimming crab and has no noticeable modifications. Being of similar size, the latter species was chosen for contrast.
Fig. 1. Metopograpsus frontalis and Thalamita sima crabs. A. Whole body of M. frontalis with black arrow indicating an opposed pair of cheliped tips and white arrow indicating a leg tip. Scale bar ¼ 10 mm. B. T. sima cheliped tip (black arrow). Scale bar ¼ 5 mm. C. Light micrograph of M. frontalis cheliped tip. D. Secondary electron image (SEI) of M. frontalis cheliped tip. E. Backscattered electron image (BSE) of M. frontalis cheliped tip: darker contrast indicates lower average atomic number. Scale bars C–E ¼ 200 mm. F. BSE of transverse section through M. frontalis cheliped tip. Outer surface is to the left. Regions on the transect line designated A, B, C, D and E are equivalent to regions in Fig. 2 designated: outer edge, near outer edge, outer middle, inner middle and inner edge respectively. G. SEI of transverse section through leg tip of M. frontalis. Outer surface is to the left. Regions on the transect line designated A, B, C and D are equivalent to regions in Fig. 2 designated: outer edge, outer middle, inner middle and inner edge respectively. H–J. X-ray intensity maps for Cl, Br and Cl þ Br respectively, across cheliped tip from area similar to that shown in boxed region of F. K–M. X-ray intensity maps for Cl, Br and Cl þ Br respectively, across leg tip from area shown in G. X-ray intensity maps use a thermal colour scale. White indicates the higher X-ray intensity for an element and black the lower; pink areas > yellow areas > green areas > blue areas. Scale bars F–I ¼ 30 mm.
B.W. Cribb et al. / Arthropod Structure & Development 38 (2009) 173–178
2.2. Scanning electron microscopy and microanalysis For secondary electron and backscattered electron imaging of whole tissues, chelipeds, legs, carapace and setae were preserved in 70% ethanol then air dried and mounted on aluminium mounts using carbon adhesive tabs. Images were collected using a JEOL 6460 low vacuum scanning electron microscope (SEM) operating at 31 Pa and 20 kV. In order to evaluate structure and composition through digestion, some cheliped tips of M. frontalis were treated with 0.9 M NaOH for 3 h at 70 C (1:10 solid to solvent ratio) with stirring, washed in distilled water 3 (overnight), immersed in 1 N HCl for 1 h at room temperature (1:15 solid to solvent ratio) with stirring and washed 2 in distilled water. These digested tissue samples were then dehydrated in ethanol to 100%, criticalpoint dried, mounted as above and platinum coated before observation at high vacuum and 8–10 kV. For microanalysis, tissue samples were hand-dried on paper towel, embedded in Daystar resin, polymerized at room temperature, sectioned and polished flat. Samples were assessed using an integrated JEOL Hyper minicup, 133 eV resolution, ultra thin window (UTW), SiLi crystal, Energy Dispersive X-ray Spectrometer (EDS). Because no suitable standard is currently available to match the material being assessed, standardless quantitative analysis was used with a phi– rho–z correction. Acquisition conditions on the SEM were 20 kV, 10 mm working distance and 30 s live time acquisition at approximately 15–20% dead time. Elemental distributions were also mapped using the same voltage. Presence of bromine was confirmed using wavelength dispersive spectroscopy on microprobe, using 20 kV, 15 nA and a 2 mm defocused beam. It should be noted that cheliped tip material sampled prior to the start of the project was harvested directly from crabs to test for the presence of chlorine, bromine and absence of calcium and zinc. Thereafter samples were harvested into 70% ethanol to avoid the possibility of bacterial degradation. Samples taken directly from crabs and taken after storage in 70% ethanol showed similar EDS spectra and samples stored in 70% ethanol did not show a progressive loss in elemental mass for Cl or Br with storage, indicating no calculable extraction of material as a result of the protocol. 2.3. Measurement of hardness (H) and reduced elastic modulus (Er) Fresh samples of cheliped and leg tip (M. frontalis) were embedded in Daystar resin and prepared as above to achieve flat cross sections. Before physical property measurements, the samples were rewet using distilled water, to avoid artificial increases in values from drying (see Scho¨berl and Jager, 2006). Flat tissue samples were indented using a Hysitron Triboindenter fitted with a Berkovich tip of tip radius 50 nm. The load function was 60 mN/s to a maximum load of 300 mN, holding for 2 s and unloading at a rate of 60 mN/s. Indents were spaced 15 mm apart. An atomic force microscope (AFM) was used to confirm that the indents were well-formed over the penetration range. Hardness (H) and reduced elastic modulus (Er) (also known as reduced Young’s modulus) were calculated using load–displacement curves by Hysitron software according to the model of Oliver and Pharr (2004). After mechanical measurement, samples were assessed for elemental composition as above using spot analysis aligned to indent points. 3. Results 3.1. Behavioural observations M. frontalis crabs were observed grazing on algae on rocky shores using their chelipeds to feed. They carry these limbs held
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high with the tips hanging downwards. As they walk side-ways over the rocks, balancing on the translucent brown spine-like leg tips, they repeatedly dip the ends of the pendant chelipeds onto the rocky substrate. Both front appendages are involved in the behaviour. At times the crabs scrape the rocky substrate with the chelipeds. Each appendage is comprised of two parts: the propodus (fixed element) and the dactylus (the more mobile medial element). The propodus is pushed against the rock while the dactylus is drawn towards it. The material scraped off the substrate is then transferred to the mouthparts. 3.2. Scanning electron microscopy and microanalysis The cheliped and leg tips of M. frontalis are tissues distinct from the remaining limb cuticle, appearing light brown and translucent (Fig. 1A). In contrast, the tips of T. sima have no similar modification and are composed of white carapace (Fig. 1B). The cheliped tips of M. frontalis are crescent-shaped and opposed (Fig. 1A,C). Under SEM using secondary electron imaging there is no difference in appearance, but when imaged with backscattered electrons (BSE) a difference in contrast appears, with the tip being darker than the carapace, which indicates a lower average atomic number for the material in the tip (contrast Fig. 1D,E). When observed using BSE, the cheliped tip shows a number of layers across a transverse section that vary in average atomic number (Fig. 1F). These have a difference in composition when mapped for elements; varying in halogen concentration (Fig. 2H–M: data presented below). When the cheliped tip is fractured across, a number of layers that differ in structure may be observed (Fig. 2A–C). Layer 1, at the outer edge and about 3 mm in depth, lies above layer 2 which is 15–20 mm thick and composed of ordered fibres (Fig. 2A,B). A third layer lies more centrally, shows no obvious orientation of fibres, and contains multiple holes in which are located perpendicular rods that penetrate the entire length of the block of non-calcified material (Fig. 3C). Rods vary in size but are about 2.5 mm in diameter. On the inner side of the transverse section lies a fourth layer. This appears poorly striated and contains fewer holes and rods than layer 3 (Fig. 2A). A fifth layer is apparent close to the inner edge (Fig. 2A). Some of these structural layers are more clearly differentiated when the material has been extracted with NaOH followed by HCl (Fig. 2D): The thin distal laminum remains (Fig. 2D: on the left); layer 2 is mostly digested (absent); and layer 3 remains intact. On the inner side layer 4 of the cheliped tip now appears crenulated with lobular fibrous regions that demonstrate a heterogeneous composition existed prior to digestion. The proximal laminum is homogeneous and 10–20 mm thick, showing an ordered fibrous nature. The leg tip is more homogeneous, with two components (Fig. 2E,F); an outer fibrous structure around an inner circular lacuna containing homogeneous material (Fig. 2E). Cheliped tips mapped for elemental distribution reveal a distinct layering in halogen mass (Fig. 1H–M) where Cl is present in highest concentrations towards the outer and inner edges and Br occurs principally in the outer region but in a more central band. The leg tips also show a distinct distribution for halogen mass with the edges and outer region containing the highest concentration of Cl and Br (Fig. 1K–M). Halogens are not detected in the central lacuna. The carapace, legs, cheliped tips and various setae of M. frontalis were assessed by point analysis for more detailed study of the composition. Table 1 shows a summary of the data for calcium (Ca) and halogens (Cl, Br). Due to the use of semiquantitative analysis, data are reported as trace (<0.05–0.5%), minor (0.5–5%) and major (>20–50%). The remaining mass comprises principally carbon (C) and oxygen (O) (not shown). Whereas spines are calcified, some setae display only minor mass % of Ca; however the leg and cheliped tips contain only trace mass % of Ca. The composition
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Fig. 2. Scanning electron micrographs of non-calcified cheliped and leg tips from Metopograpsus frontalis, in transverse section. A. Fractured surface across a cheliped tip showing multiple layers. Outer surface is to the left. Scale bar ¼ 20 mm. B. Fractured surface across a cheliped tip showing layers 1–3. Outer surface is to the left. Scale bar ¼ 10 mm. C. Fractured surface of a cheliped tip showing rods (r) filling and protruding from holes in layer 3 region. D. Transverse section through the cheliped tip extracted with NaOH followed by HCl. Outer surface is to the left. White double-headed arrow indicates lobe on crenulated inner surface. Numbers indicate layers. E. Partially fractured surface across a leg tip: ofs, outer fibrous structure; icl inner circular lacuna. Outer surface is to the left. F. Enlarged area of fractured leg tip. Scale bar ¼ 10 mm.
of the carapace, measured on the chelipeds of T. sima was also assessed for comparison. For T. sima (n ¼ 9), the carapace contains trace amounts of Cl (0.24 0.06) and Br (0.16 0.05) and major amounts of Ca (43.02 2.04). 3.3. Measurement of hardness (H) and reduced elastic modulus (Er) The carapace of M. frontalis is significantly harder than the cheliped tip and leg tip, both of which have a similar hardness (Fig. 3A) (Kruskal–Wallis: H ¼ 16.21, df ¼ 1, P < 0.001). Similar
results were obtained for Er (Fig. 3B) (Kruskal–Wallis: H ¼ 49.27, df ¼ 3, P < 0.001). The H and Er were measured at regular intervals (15 mm) within the cross section of cheliped and leg tip from outer to inner side and data matched for region. Neither H nor Er differs significantly for the cheliped or the leg (Fig. 3C–F). Data are normally distributed so one-way analysis of variance was used for analysis [for cheliped tip H against region: F 0.64 df ¼ 4 P ¼ 0.639; for cheliped tip Er against region: F 1.99 df ¼ 4 P ¼ 0.117; for leg tip H against region: F 0.40 df ¼ 3 P ¼ 0.756; for leg tip Er against region: F 1.96 df ¼ 3 P ¼ 0.146]. To assess the relationship between either H or
B.W. Cribb et al. / Arthropod Structure & Development 38 (2009) 173–178
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Fig. 3. Physical measurements taken for Metopograpsus frontalis. A. Mean hardness (H) (GPa SEM) and B. Mean reduced elastic modulus (Er) (GPa SEM). Letters that differ indicate significant difference (multiple comparisons of means following Kruskal–Wallis test). C. Mean H (GPa SEM) for cheliped tips; n ¼ 8. D. Mean Er (GPa SEM) for cheliped tips; n ¼ 8. E. Mean H (GPa SEM) for leg tips; n ¼ 7. F. Mean Er (GPa SEM) for leg tips; n ¼ 7. Regions in transects were matched for size and positions. G. Scatter plot of H (GPa) against Cl mass% for all positions across a transect for cheliped tips. H. Scatter plot of H (GPa) against Cl mass% for matched sections at 30 mm from the outer side of the cheliped tips.
Er and halogen mass % in individual crabs, data were plotted as scatter graphs for Cl, Br and total halogen (Cl þ Br) mass%. No correlative relationship is apparent for any comparisons (examples: Fig. 3G,H). Table 1 Average dry weight as mass% ( SEM) of elements, Ca, Cl and Br, for Metopograpsus frontalis crabs, determined using standardless quantitative EDS and a phi–rho–z matrix correction (carapace n ¼ 4, spines n ¼ 8, setae n ¼ 8, leg tips n ¼ 10, cheliped tips n ¼ 10). Structure
Cl
Br
Ca
Carapace Spines Setae Leg tips Cheliped tips
Trace (0.18 0.1) Trace (<0.05) Trace (0.15 0.1) Minor (3.73 0.2) Minor (3.85 0.2)
Nil (0 0) Nil (0 0) Minor (0.66 0.3) Minor (1.63 0.4) Minor (2.28 0.4)
Major (41.91 0.2) Major (25.12 0.2) Minor (0.71 0.3) Trace (<0.05) Trace (<0.05)
4. Discussion Previously, Schofield et al. (2003) reported that as Ca concentrations drop, the predominant inorganic element in the tips of tarsal claws in crabs becomes Cl. In contrast, unpublished data from Schofield and Nesson on the shore crab, Pachygrapsus crassipes (reported in Schofield, 2005) indicated composition of cheliped tips as containing bromine. We have addressed in specific, the structure and composition of bulk non-calcified exoskeleton found in the grapsid shore crab M. frontalis. This material is found at the tips of legs and chelipeds. It is recognisable from its light brown colour and translucent appearance; however it is not merely cuticle with the calcium removed. This non-calcified exoskeleton contains significant concentrations of both Cl and Br. In comparison, body setae have slightly higher Ca mass and show lower concentrations of
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halogens, suggesting that a range of intermediate forms of exoskeleton may exist. Carapace, with the highest Ca mass has only trace Cl and no Br in M. frontalis. Similar levels of Ca and halogens were found for the carapace of T. sima which lacks the non-calcified exoskeleton at the cheliped and leg tips. So it can be stated that there is no specific compositional adaptation of the carapace in M. frontalis that might predispose development of bulk non-calcified tissue. The structure of the cheliped tip shows a composite hierarchy. As well as layers across the tissue, there are rods that travel longitudinally through the tissue, perhaps reinforcing it. In contrast the leg tips are less complex. Instead of individual rods there is a central channel. However both tissues show overall similarity in distribution of halogens, with a region on the inner side that is low in halogen concentration. This hints at the cheliped tip being a leg tip, modified for rasping and grasping. Also, in terms of physical properties, hardness and reduced elastic modulus were similar for both leg and cheliped tips, as well as across the tissue transects. The values, while lower than those found for the calcified tissues, were equivalent to values found for sclerotised insect cuticle that lacks metals: H 0.24–0.78 GPa; E 1–20 GPa, in sclerotised cuticles (see Vincent and Wegst, 2004). One rider which must be applied to the data obtained for H and Er is that the measurements were taken in the direction of delamination of the cuticle layers rather than perpendicular to the cuticle surface, however since the material is subject to use and wear in this cross-sectional aspect it can be considered the biologically relevant direction. Previous studies have shown that exoskeletons of some marine polychaete worms and non-calcified arthropods such as insects show a correlation between hardness and metals that co-locate with halogens (Schofield et al., 2002, 2003; Lichtenegger et al., 2003; Birkedal et al., 2006; Cribb et al., 2008). These links likely involve the metal and halogen binding with proteins (Schofield, 2001; Lichtenegger et al., 2003; Birkedal et al., 2006). The ratio of metal to halogen appears to vary between organisms (Cribb et al., 2008). Birkedal et al. (2006) have shown the presence of a number of post-translationally modified amino acids in the jaws of Nereis. These are multiply halogenated by chlorine, bromine, and/or iodine. They concluded that while bromine and iodine are unlikely to play a purely mechanical role, the local zinc and chlorine concentrations and jaw microstructure are the prime determinants of local jaw hardness. Cuticle that is enriched with halogens, in which metals are absent or only present at trace levels, has not yet been studied on its own. Khan et al. (2006) found that in the jaws of Nereis, while Br and I were present with single chemical environments, Cl was present in two modes, Cl–Zn and Cl–C, with the latter comprising 40% of the Cl content. This points to at least one halogen possibly forming an important structural component in the absence of a metal. The exoskeleton of M. frontalis has provided a useful model to examine the correlation of halogens with physical properties of hardness and elastic modulus within a chitin-based matrix in the absence of metals. Neither Cl nor Br concentration were found to correlate with H and/or Er in this tissue type. These data suggest that halogens, and the forms in which they occur in the absence of metals, do not correlate specifically with harder or stiffer cuticle.
The question remains as to what advantage this non-calcified material may confer. Instead of differences in H and Er, the role of non-calcified tissues may be functionally related to friction or wear resistance. Khan et al. (2006) found that halogens are scarce at the tip of the jaws of Nereis, where wear resistance properties are most needed, while present in high concentrations at the base. From these data they argued that halogens are not contributors to properties of wear and hardness. However the unpublished, and perhaps more directly relevant work of Schofield and Nesson (as reported in Schofield, 2005) stated that cheliped tips of P. crassipes are more resistant to ‘‘chipping’’ than the surrounding calcified tissue, when exposed to bead blasting, suggesting some improvements in wear. Other physical properties still need to be explored, for example the possible role of increased friction, which might enhance grip when compared with calcified tissues. This is likely to be important in grasping crabs.
Acknowledgements We thank Dr Peter Davie from the Queensland Museum who confirmed identification of the crabs used in the study.
References Birkedal, H., Khan, R.K., Slack, N., Broomell, C., Lichtenegger, H.C., Zok, F., Stucky, G.D., Waite, J.H., 2006. Halogenated veneers: protein cross-linking and halogenation in the jaws of Nereis, a marine polychaete worm. Chembiochem 7, 1392–1399. Compe`re, P., Goffinet, G., 1987. Aspects ultrastructuraux et fonctionnels de diverses regions cuticulaires non mine´ralise´es d’un crustace´ de´capode, Carcinus maenas. Annals de la Society Royale Zoologique de Belgique 117, 159–173. Cribb, B.W., Stewart, A., Huang, H., Truss, R., Noller, B., Rasch, R., Zalucki, M.P., 2008. Insect mandibles – comparative mechanical properties and links with metal incorporation. Naturwissenschaften 95, 17–23. Khan, R.K., Stoimenov, P.K., Mates, T.E., Waite, J.H., Stucky, G.D., 2006. Exploring gradients of halogens and zinc in the surface and subsurface of Nereis jaws. Langmuir 22, 8465–8470. Lichtenegger, H.C., Schoeberl, T., Ruokolainen, J.T., Cross, J.O., Heald, S.M., Birkedal, H., Waite, J.H., Stucky, G.D., 2003. Zinc and mechanical prowess in the jaws of Nereis, a marine worm. Proceedings of the National Academy of Sciences of the United States of America 100, 9144–9149. Oliver, W.C., Pharr, G.M., 2004. Measurement of hardness and elastic modulus by instrumented indentation: advances in understanding and refinements to methodology. Journal of Materials Research 19, 3–20. Scho¨berl, T., Jager, I.L., 2006. Wet or dry – hardness, stiffness and wear resistance of biological materials on the micron scale. Advanced Engineering Materials 8, 1164–1169. Schofield, R.M.S., 2001. Metals in cuticular structures. In: Brownell, P., Polis, G. (Eds.), Scorpion Biology and Research. Oxford University Press, Oxford, U.K, pp. 234–256. Schofield, R.M.S., 2005. Metal–halogen biomaterials. American Entomologist 51, 45–47. Schofield, R.M.S., Nesson, M.H., Richardson, K.A., 2002. Tooth hardness increases with zinc-content in mandibles of young adult leaf-cutter ants. Naturwissenschaften 89, 579–583. Schofield, R.M.S., Nesson, M.H., Richardson, K.A., Wyeth, P., 2003. Zinc is incorporated into cuticular ‘‘tools’’ after ecdysis: the time course of the zinc distribution in ‘‘tools’’ and whole bodies of an ant and a scorpion. Journal of Insect Physiology 49, 31–44. Shaw, M., Tibbetts, I.R., 2004. Grazing by Metopograpsus frontalis (Decapoda: Grapsidae) on intertidal rock walls of Moreton Bay. Proceedings of the Royal Society of Queensland 111, 95–101. Vincent, J.F.V., Wegst, U.G.K., 2004. Design and mechanical properties of insect cuticle. Arthropod Structure and Development 33, 187–199.
Contents lists available at ScienceDirect
Arthropod Structure & Development journal homepage: www.elsevier.com/locate/asd
Structure, composition and properties of naturally occurring non-calcified crustacean cuticle B.W. Cribb a, b, *, A. Rathmell a, b, R. Charters a, b, R. Rasch a, H. Huang c, I.R. Tibbetts d a
The University of Queensland, Centre for Microscopy & Microanalysis, Brisbane 4072, Australia The University of Queensland, School of Integrative Biology, Brisbane 4072, Australia c The University of Queensland, School of Engineering, Brisbane 4072, Australia d The University of Queensland, Centre for Marine Studies, Brisbane 4072, Australia b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 September 2008 Accepted 13 November 2008
Crustaceans are known for their hard, calcified exoskeleton; however some regions appear different in colour and opacity. These include leg and cheliped tips in the grapsid crab, Metopograpsus frontalis. The chelipeds and leg tips contain only trace levels of calcium but a significant mass of the halogens, chlorine (Cl) and bromine (Br). In contrast, the carapace is heavily calcified and contains only a trace mass of Cl and no Br. In transverse section across the non-calcified tip regions of cheliped and leg the mass percent of halogens varies with position. As such, the exoskeleton of M. frontalis provides a useful model to examine a possible correlation of halogen concentration with the physical properties of hardness (H) and reduced elastic modulus (Er), within a chitin-based matrix. Previously published work suggests a correlation exists between Cl concentration and hardness in similar tissues that contain a metal (e.g. zinc). However, in M. frontalis H and Er did not vary significantly across cheliped or leg tip despite differences in halogen concentration. The non-calcified regions of M. frontalis are less hard and less stiff than the carapace but equivalent to values found for insect cuticle lacking metals. Cheliped tips showed a complex morphological layering that differed from leg tips. Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Chitin Polysaccharides Stiffness Nanoindentation Arthropod Crab
1. Introduction Crustaceans are well known for their hard, calcified exoskeleton, but some regions show flexibility, indicating a different structure and composition. Surprisingly little is known about these alternative cuticles. A morphological study has been carried out on intersegmental membranes, branchial and inner branchiostegal cuticles of the crab, Carcinus maenas (Compe`re and Goffinet, 1987). But these are not the only regions that appear to be non-calcified in crustaceans. The leg tips and grasping claws (cheliped tips) of grapsid crabs are of a different colour and opacity from the carapace and are rubbery to touch; characters potentially indicative of a lack of typical mineralization. Hints about composition are available from unpublished findings reported by Schofield et al. (2003) but nothing has been formally published on structure, composition, hardness or stiffness of these materials. Schofield et al. (2003) indicate that as calcium
* Corresponding author. The University of Queensland, Centre for Microscopy & Microanalysis, St. Lucia Campus, Brisbane 4072, Australia. Tel.: þ61 7 33657086; fax: þ61 7 3346 3993. E-mail address: [email protected] (B.W. Cribb). 1467-8039/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.asd.2008.11.002
levels drop, the predominant inorganic element in the tips of tarsal claws in crabs becomes chlorine (Cl). The grapsid, or grasping crab, Metopograpsus frontalis uses the tips of the chelipeds to scrape and tug algae from intertidal substrate surfaces which it then eats (Shaw and Tibbetts, 2004). These chelipeds and legs are tipped with a translucent light brown coloured material that is readily distinguished from the rest of the carapace. The current study examines the structure, composition and the physical properties of hardness and elastic modulus for this unusual material. 2. Materials and methods 2.1. Animals and behavioural observations Two species of crab, M. frontalis (Grapsidae) and Thalamita sima (Portunidae), were collected from sites around Moreton Bay, Queensland, Australia (Fig. 1A, B). While M. frontalis is a grapsid crab and shows modification of the tips of the chelipeds and legs (Fig. 1A), T. sima is a portunid or swimming crab and has no noticeable modifications. Being of similar size, the latter species was chosen for contrast.
Fig. 1. Metopograpsus frontalis and Thalamita sima crabs. A. Whole body of M. frontalis with black arrow indicating an opposed pair of cheliped tips and white arrow indicating a leg tip. Scale bar ¼ 10 mm. B. T. sima cheliped tip (black arrow). Scale bar ¼ 5 mm. C. Light micrograph of M. frontalis cheliped tip. D. Secondary electron image (SEI) of M. frontalis cheliped tip. E. Backscattered electron image (BSE) of M. frontalis cheliped tip: darker contrast indicates lower average atomic number. Scale bars C–E ¼ 200 mm. F. BSE of transverse section through M. frontalis cheliped tip. Outer surface is to the left. Regions on the transect line designated A, B, C, D and E are equivalent to regions in Fig. 2 designated: outer edge, near outer edge, outer middle, inner middle and inner edge respectively. G. SEI of transverse section through leg tip of M. frontalis. Outer surface is to the left. Regions on the transect line designated A, B, C and D are equivalent to regions in Fig. 2 designated: outer edge, outer middle, inner middle and inner edge respectively. H–J. X-ray intensity maps for Cl, Br and Cl þ Br respectively, across cheliped tip from area similar to that shown in boxed region of F. K–M. X-ray intensity maps for Cl, Br and Cl þ Br respectively, across leg tip from area shown in G. X-ray intensity maps use a thermal colour scale. White indicates the higher X-ray intensity for an element and black the lower; pink areas > yellow areas > green areas > blue areas. Scale bars F–I ¼ 30 mm.
B.W. Cribb et al. / Arthropod Structure & Development 38 (2009) 173–178
2.2. Scanning electron microscopy and microanalysis For secondary electron and backscattered electron imaging of whole tissues, chelipeds, legs, carapace and setae were preserved in 70% ethanol then air dried and mounted on aluminium mounts using carbon adhesive tabs. Images were collected using a JEOL 6460 low vacuum scanning electron microscope (SEM) operating at 31 Pa and 20 kV. In order to evaluate structure and composition through digestion, some cheliped tips of M. frontalis were treated with 0.9 M NaOH for 3 h at 70 C (1:10 solid to solvent ratio) with stirring, washed in distilled water 3 (overnight), immersed in 1 N HCl for 1 h at room temperature (1:15 solid to solvent ratio) with stirring and washed 2 in distilled water. These digested tissue samples were then dehydrated in ethanol to 100%, criticalpoint dried, mounted as above and platinum coated before observation at high vacuum and 8–10 kV. For microanalysis, tissue samples were hand-dried on paper towel, embedded in Daystar resin, polymerized at room temperature, sectioned and polished flat. Samples were assessed using an integrated JEOL Hyper minicup, 133 eV resolution, ultra thin window (UTW), SiLi crystal, Energy Dispersive X-ray Spectrometer (EDS). Because no suitable standard is currently available to match the material being assessed, standardless quantitative analysis was used with a phi– rho–z correction. Acquisition conditions on the SEM were 20 kV, 10 mm working distance and 30 s live time acquisition at approximately 15–20% dead time. Elemental distributions were also mapped using the same voltage. Presence of bromine was confirmed using wavelength dispersive spectroscopy on microprobe, using 20 kV, 15 nA and a 2 mm defocused beam. It should be noted that cheliped tip material sampled prior to the start of the project was harvested directly from crabs to test for the presence of chlorine, bromine and absence of calcium and zinc. Thereafter samples were harvested into 70% ethanol to avoid the possibility of bacterial degradation. Samples taken directly from crabs and taken after storage in 70% ethanol showed similar EDS spectra and samples stored in 70% ethanol did not show a progressive loss in elemental mass for Cl or Br with storage, indicating no calculable extraction of material as a result of the protocol. 2.3. Measurement of hardness (H) and reduced elastic modulus (Er) Fresh samples of cheliped and leg tip (M. frontalis) were embedded in Daystar resin and prepared as above to achieve flat cross sections. Before physical property measurements, the samples were rewet using distilled water, to avoid artificial increases in values from drying (see Scho¨berl and Jager, 2006). Flat tissue samples were indented using a Hysitron Triboindenter fitted with a Berkovich tip of tip radius 50 nm. The load function was 60 mN/s to a maximum load of 300 mN, holding for 2 s and unloading at a rate of 60 mN/s. Indents were spaced 15 mm apart. An atomic force microscope (AFM) was used to confirm that the indents were well-formed over the penetration range. Hardness (H) and reduced elastic modulus (Er) (also known as reduced Young’s modulus) were calculated using load–displacement curves by Hysitron software according to the model of Oliver and Pharr (2004). After mechanical measurement, samples were assessed for elemental composition as above using spot analysis aligned to indent points. 3. Results 3.1. Behavioural observations M. frontalis crabs were observed grazing on algae on rocky shores using their chelipeds to feed. They carry these limbs held
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high with the tips hanging downwards. As they walk side-ways over the rocks, balancing on the translucent brown spine-like leg tips, they repeatedly dip the ends of the pendant chelipeds onto the rocky substrate. Both front appendages are involved in the behaviour. At times the crabs scrape the rocky substrate with the chelipeds. Each appendage is comprised of two parts: the propodus (fixed element) and the dactylus (the more mobile medial element). The propodus is pushed against the rock while the dactylus is drawn towards it. The material scraped off the substrate is then transferred to the mouthparts. 3.2. Scanning electron microscopy and microanalysis The cheliped and leg tips of M. frontalis are tissues distinct from the remaining limb cuticle, appearing light brown and translucent (Fig. 1A). In contrast, the tips of T. sima have no similar modification and are composed of white carapace (Fig. 1B). The cheliped tips of M. frontalis are crescent-shaped and opposed (Fig. 1A,C). Under SEM using secondary electron imaging there is no difference in appearance, but when imaged with backscattered electrons (BSE) a difference in contrast appears, with the tip being darker than the carapace, which indicates a lower average atomic number for the material in the tip (contrast Fig. 1D,E). When observed using BSE, the cheliped tip shows a number of layers across a transverse section that vary in average atomic number (Fig. 1F). These have a difference in composition when mapped for elements; varying in halogen concentration (Fig. 2H–M: data presented below). When the cheliped tip is fractured across, a number of layers that differ in structure may be observed (Fig. 2A–C). Layer 1, at the outer edge and about 3 mm in depth, lies above layer 2 which is 15–20 mm thick and composed of ordered fibres (Fig. 2A,B). A third layer lies more centrally, shows no obvious orientation of fibres, and contains multiple holes in which are located perpendicular rods that penetrate the entire length of the block of non-calcified material (Fig. 3C). Rods vary in size but are about 2.5 mm in diameter. On the inner side of the transverse section lies a fourth layer. This appears poorly striated and contains fewer holes and rods than layer 3 (Fig. 2A). A fifth layer is apparent close to the inner edge (Fig. 2A). Some of these structural layers are more clearly differentiated when the material has been extracted with NaOH followed by HCl (Fig. 2D): The thin distal laminum remains (Fig. 2D: on the left); layer 2 is mostly digested (absent); and layer 3 remains intact. On the inner side layer 4 of the cheliped tip now appears crenulated with lobular fibrous regions that demonstrate a heterogeneous composition existed prior to digestion. The proximal laminum is homogeneous and 10–20 mm thick, showing an ordered fibrous nature. The leg tip is more homogeneous, with two components (Fig. 2E,F); an outer fibrous structure around an inner circular lacuna containing homogeneous material (Fig. 2E). Cheliped tips mapped for elemental distribution reveal a distinct layering in halogen mass (Fig. 1H–M) where Cl is present in highest concentrations towards the outer and inner edges and Br occurs principally in the outer region but in a more central band. The leg tips also show a distinct distribution for halogen mass with the edges and outer region containing the highest concentration of Cl and Br (Fig. 1K–M). Halogens are not detected in the central lacuna. The carapace, legs, cheliped tips and various setae of M. frontalis were assessed by point analysis for more detailed study of the composition. Table 1 shows a summary of the data for calcium (Ca) and halogens (Cl, Br). Due to the use of semiquantitative analysis, data are reported as trace (<0.05–0.5%), minor (0.5–5%) and major (>20–50%). The remaining mass comprises principally carbon (C) and oxygen (O) (not shown). Whereas spines are calcified, some setae display only minor mass % of Ca; however the leg and cheliped tips contain only trace mass % of Ca. The composition
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Fig. 2. Scanning electron micrographs of non-calcified cheliped and leg tips from Metopograpsus frontalis, in transverse section. A. Fractured surface across a cheliped tip showing multiple layers. Outer surface is to the left. Scale bar ¼ 20 mm. B. Fractured surface across a cheliped tip showing layers 1–3. Outer surface is to the left. Scale bar ¼ 10 mm. C. Fractured surface of a cheliped tip showing rods (r) filling and protruding from holes in layer 3 region. D. Transverse section through the cheliped tip extracted with NaOH followed by HCl. Outer surface is to the left. White double-headed arrow indicates lobe on crenulated inner surface. Numbers indicate layers. E. Partially fractured surface across a leg tip: ofs, outer fibrous structure; icl inner circular lacuna. Outer surface is to the left. F. Enlarged area of fractured leg tip. Scale bar ¼ 10 mm.
of the carapace, measured on the chelipeds of T. sima was also assessed for comparison. For T. sima (n ¼ 9), the carapace contains trace amounts of Cl (0.24 0.06) and Br (0.16 0.05) and major amounts of Ca (43.02 2.04). 3.3. Measurement of hardness (H) and reduced elastic modulus (Er) The carapace of M. frontalis is significantly harder than the cheliped tip and leg tip, both of which have a similar hardness (Fig. 3A) (Kruskal–Wallis: H ¼ 16.21, df ¼ 1, P < 0.001). Similar
results were obtained for Er (Fig. 3B) (Kruskal–Wallis: H ¼ 49.27, df ¼ 3, P < 0.001). The H and Er were measured at regular intervals (15 mm) within the cross section of cheliped and leg tip from outer to inner side and data matched for region. Neither H nor Er differs significantly for the cheliped or the leg (Fig. 3C–F). Data are normally distributed so one-way analysis of variance was used for analysis [for cheliped tip H against region: F 0.64 df ¼ 4 P ¼ 0.639; for cheliped tip Er against region: F 1.99 df ¼ 4 P ¼ 0.117; for leg tip H against region: F 0.40 df ¼ 3 P ¼ 0.756; for leg tip Er against region: F 1.96 df ¼ 3 P ¼ 0.146]. To assess the relationship between either H or
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Fig. 3. Physical measurements taken for Metopograpsus frontalis. A. Mean hardness (H) (GPa SEM) and B. Mean reduced elastic modulus (Er) (GPa SEM). Letters that differ indicate significant difference (multiple comparisons of means following Kruskal–Wallis test). C. Mean H (GPa SEM) for cheliped tips; n ¼ 8. D. Mean Er (GPa SEM) for cheliped tips; n ¼ 8. E. Mean H (GPa SEM) for leg tips; n ¼ 7. F. Mean Er (GPa SEM) for leg tips; n ¼ 7. Regions in transects were matched for size and positions. G. Scatter plot of H (GPa) against Cl mass% for all positions across a transect for cheliped tips. H. Scatter plot of H (GPa) against Cl mass% for matched sections at 30 mm from the outer side of the cheliped tips.
Er and halogen mass % in individual crabs, data were plotted as scatter graphs for Cl, Br and total halogen (Cl þ Br) mass%. No correlative relationship is apparent for any comparisons (examples: Fig. 3G,H). Table 1 Average dry weight as mass% ( SEM) of elements, Ca, Cl and Br, for Metopograpsus frontalis crabs, determined using standardless quantitative EDS and a phi–rho–z matrix correction (carapace n ¼ 4, spines n ¼ 8, setae n ¼ 8, leg tips n ¼ 10, cheliped tips n ¼ 10). Structure
Cl
Br
Ca
Carapace Spines Setae Leg tips Cheliped tips
Trace (0.18 0.1) Trace (<0.05) Trace (0.15 0.1) Minor (3.73 0.2) Minor (3.85 0.2)
Nil (0 0) Nil (0 0) Minor (0.66 0.3) Minor (1.63 0.4) Minor (2.28 0.4)
Major (41.91 0.2) Major (25.12 0.2) Minor (0.71 0.3) Trace (<0.05) Trace (<0.05)
4. Discussion Previously, Schofield et al. (2003) reported that as Ca concentrations drop, the predominant inorganic element in the tips of tarsal claws in crabs becomes Cl. In contrast, unpublished data from Schofield and Nesson on the shore crab, Pachygrapsus crassipes (reported in Schofield, 2005) indicated composition of cheliped tips as containing bromine. We have addressed in specific, the structure and composition of bulk non-calcified exoskeleton found in the grapsid shore crab M. frontalis. This material is found at the tips of legs and chelipeds. It is recognisable from its light brown colour and translucent appearance; however it is not merely cuticle with the calcium removed. This non-calcified exoskeleton contains significant concentrations of both Cl and Br. In comparison, body setae have slightly higher Ca mass and show lower concentrations of
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halogens, suggesting that a range of intermediate forms of exoskeleton may exist. Carapace, with the highest Ca mass has only trace Cl and no Br in M. frontalis. Similar levels of Ca and halogens were found for the carapace of T. sima which lacks the non-calcified exoskeleton at the cheliped and leg tips. So it can be stated that there is no specific compositional adaptation of the carapace in M. frontalis that might predispose development of bulk non-calcified tissue. The structure of the cheliped tip shows a composite hierarchy. As well as layers across the tissue, there are rods that travel longitudinally through the tissue, perhaps reinforcing it. In contrast the leg tips are less complex. Instead of individual rods there is a central channel. However both tissues show overall similarity in distribution of halogens, with a region on the inner side that is low in halogen concentration. This hints at the cheliped tip being a leg tip, modified for rasping and grasping. Also, in terms of physical properties, hardness and reduced elastic modulus were similar for both leg and cheliped tips, as well as across the tissue transects. The values, while lower than those found for the calcified tissues, were equivalent to values found for sclerotised insect cuticle that lacks metals: H 0.24–0.78 GPa; E 1–20 GPa, in sclerotised cuticles (see Vincent and Wegst, 2004). One rider which must be applied to the data obtained for H and Er is that the measurements were taken in the direction of delamination of the cuticle layers rather than perpendicular to the cuticle surface, however since the material is subject to use and wear in this cross-sectional aspect it can be considered the biologically relevant direction. Previous studies have shown that exoskeletons of some marine polychaete worms and non-calcified arthropods such as insects show a correlation between hardness and metals that co-locate with halogens (Schofield et al., 2002, 2003; Lichtenegger et al., 2003; Birkedal et al., 2006; Cribb et al., 2008). These links likely involve the metal and halogen binding with proteins (Schofield, 2001; Lichtenegger et al., 2003; Birkedal et al., 2006). The ratio of metal to halogen appears to vary between organisms (Cribb et al., 2008). Birkedal et al. (2006) have shown the presence of a number of post-translationally modified amino acids in the jaws of Nereis. These are multiply halogenated by chlorine, bromine, and/or iodine. They concluded that while bromine and iodine are unlikely to play a purely mechanical role, the local zinc and chlorine concentrations and jaw microstructure are the prime determinants of local jaw hardness. Cuticle that is enriched with halogens, in which metals are absent or only present at trace levels, has not yet been studied on its own. Khan et al. (2006) found that in the jaws of Nereis, while Br and I were present with single chemical environments, Cl was present in two modes, Cl–Zn and Cl–C, with the latter comprising 40% of the Cl content. This points to at least one halogen possibly forming an important structural component in the absence of a metal. The exoskeleton of M. frontalis has provided a useful model to examine the correlation of halogens with physical properties of hardness and elastic modulus within a chitin-based matrix in the absence of metals. Neither Cl nor Br concentration were found to correlate with H and/or Er in this tissue type. These data suggest that halogens, and the forms in which they occur in the absence of metals, do not correlate specifically with harder or stiffer cuticle.
The question remains as to what advantage this non-calcified material may confer. Instead of differences in H and Er, the role of non-calcified tissues may be functionally related to friction or wear resistance. Khan et al. (2006) found that halogens are scarce at the tip of the jaws of Nereis, where wear resistance properties are most needed, while present in high concentrations at the base. From these data they argued that halogens are not contributors to properties of wear and hardness. However the unpublished, and perhaps more directly relevant work of Schofield and Nesson (as reported in Schofield, 2005) stated that cheliped tips of P. crassipes are more resistant to ‘‘chipping’’ than the surrounding calcified tissue, when exposed to bead blasting, suggesting some improvements in wear. Other physical properties still need to be explored, for example the possible role of increased friction, which might enhance grip when compared with calcified tissues. This is likely to be important in grasping crabs.
Acknowledgements We thank Dr Peter Davie from the Queensland Museum who confirmed identification of the crabs used in the study.
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