- Email: [email protected]
Structure and composition of the aragonitic crossed lamellar layers in six species of Bivalvia and Gastropoda
Comparative Biochemistry and Physiology Part A 126 (2000) 367 – 377
www.elsevier.com/locate/cbpa
Structure and composition of the aragonitic crossed lamellar layers in six species of Bivalvia and Gastropoda Y. Dauphin *, A. Denis UMR 8616, Laboratoire de Pale´ontologie, Uni6ersite´ Paris XI, Baˆt. 504, Orsay F-91405, France Received 25 January 2000; received in revised form 3 May 2000; accepted 9 May 2000
Abstract The microstructures, the chemical composition and the soluble organic matrices of the aragonitic crossed lamellar layers of the shells of six species of molluscs have been studied. The microstructures and chemical contents are similar, whereas the quantities of organic matrices are variable. All the soluble matrices are glycoproteins, with low S contents. Their molecular weights, the protein-sugar ratios and acidities are variable. Neither a gastropod nor a bivalve pattern is recognized. The diversity of the organic matrices probably plays a main role in the fossilization processes of mollusc shells. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Biomineralization; Bivalvia; Gastropoda; Crossed lamellar layer; Aragonite; Skeletal organic matrices; DRIFT; HPLC
1. Introduction Shells of the three major mollusc classes: Cephalopoda, Gastropoda and Bivalvia are characteristically layered structures. They are composed of calcium carbonate (aragonite and/or calcite) and organic material. Bøggild (1930) recognized the main microstructural types and the distributions of calcite and aragonite. Among them, the aragonitic nacreous layer has been one of the most intensively studied and provides the basic data to elaborate some models of biomineralization. However, the simple geometric of the nacreous layer and the abundance of its organic matrix do not reflect the complexity of other microstructural types. * Corresponding author. Tel.: + 33-1-69156117; fax: +331-69156123. E-mail address: [email protected] (Y. Dauphin).
The crossed lamellar layer is the most widespread in Gastropoda and Bivalvia. Except Patella, it is aragonitic. The arrangement of aragonitic crystallites was described by Bøggild (1930), then detailed by several authors (Kobayashi 1964, 1966, 1969; McClintock, 1967; Taylor et al., 1969, 1973; Wise 1971; Cuif et al., 1985; Kobayashi, 1994). According to Wilmot et al. (1992), the basic structure of the crossed-lamellar layer is similar in all the studied species. Differential thermal analyses of several Bivalvia shells show that the organic matrix of the crossed lamellar layers differs from those of the prismatic layers (Kobayashi, 1973). Uozumi et al. (1972) described the third order lamellae surrounded by an organic matrix (conchyolin). Weiner et al. (1977) have shown that the protein fraction of the soluble matrices of crossed lamellar layers of some molluscs have variably non-discrete molecular weights. The crossed lamellar layer of Strombus is rich in acidic amino-acids (aspartic and glutamic
1095-6433/00/$ - see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 1 0 9 5 - 6 4 3 3 ( 0 0 ) 0 0 2 1 3 - 0
368
Y. Dauphin, A. Denis / Comparati6e Biochemistry and Physiology, Part A 126 (2000) 367–377
acids) (Nakahara et al., 1981). Samata (1990) confirmed the highly acidic composition of the soluble matrix. More recently, Kobayashi (1994) described a probable organic matrix inside the crystals of the crossed lamellar layer of Glycymeris. However, very little is known about the composition of the organic matrix in these layers. In this paper, we compare the mineral and organic components of the aragonitic crossed lamellar layers in six species of molluscs (three Gastropoda and three Bivalvia).
surements were made using a live time of 100 or 200 s. The electron accelerating voltage applied to the electron gun was 15 kV. The electron beam had a diameter of 100 nm. The elements Na, Mg, Al, P, S, K, Ca, Mn, Fe and Sr were selected to illustrate aspects of shell composition. Several (\ 10) microprobe analyses were made at various locations on each sample. These punctual analyses have been averaged to obtain an ‘individual’ mean. Some elements are below the detection limit of the microprobe (i.e. Mg, Fe). However, they are accounted because their low levels show a good preservation of the shells.
2. Material and methods
2.1. Material Gastropoda: Prosobranchia Cypraea le6iathan Burgess and Arnelle, 1981 (French Polynesia, Tuamotu Islands); Phalium granulatum Born, 1778, unknown origin; Strombus gigas Linne´ 1758 (Caraibes: Martinique, Guadeloupe). Bivalvia: Heterodonta Tridacna sp., unknown origin; Dosinia ponderosa (Gray, 1838) from South California; Cardium sp., unknown origin.
2.2. Methods 2.2.1. Scanning electron microscope (SEM) Fractures and polished etched sections have been observed with Philips 505 and XL30 SEM. Acidic etchings were used to reveal the details of the microstructures of the polished sections. Details of the etchings are given in the figure captions. 2.2.2. Chemical analysis Energy dispersive X-ray microanalysis (EDS) was done using a Philips 505 SEM equipped with a solid state X-ray detector. Quantitative analysis was done on shells by using the Link AN 10 000 ZAF/PB program which estimates peak-to-background ratios. The ZAF/PB method can be applied to bulk specimens with rough surfaces. Small pieces of shells were embedded in an epoxy resin and polished using various grades of diamond paste. A light etching (formic acid, 5%, 15 s) of the polished surfaces was made to reveal the structural details of the samples. The positions of the analysed points with respect to the structural features are, therefore, precisely known. A cobalt sample was used to provide the calibration. Mea-
2.2.3. FTIR Infrared analyses have been carried out on powdered shells. All spectra were recorded at 4 cm − 1 resolution with 64 scans (measurement time \ 4 min) with a string Norton–Beer apodization on a Perkin–Elmer Model 1600 Fourier transform infra-red spectrometer (FTIR), from 4000 to 450 cm − 1. The spectrometer was equipped with a diffuse reflectance accessory, that permits DRIFT measurements with high sensitivity on powders. All spectra were corrected by the Kubelka–Munk function. Before a sample spectra were run, a background spectrum was measured for pure KBr. Sample spectra were automatically ratioed against background to reduce CO2 and H2O bands. They were dried in an oven at 38°C for 1 night. Powdered shells and KBr were mixed (: 5% powdered shell in KBr) and loaded in the sample cup. The mineralogy and composition of shells can be obtained by infrared analysis, even if this method is not usual for such samples. The infrared spectrum of aragonite is characterized by three prominent absorption maxima at 1470–1490 cm − 1 (n3), 858–844 cm − 1 (n2) and 712–700 cm − 1 (n4). Bands of lesser intensity appear at 1785, 1130 and 1060–1080 cm − 1 (n1). From spectra between 2000 and 400 cm − 1, White (1974) concluded that ‘‘The organically derived materials are spectroscopically indistinguishable from inorganic materials’’. However, reexamination of recent and fossil invertebrate shells with a larger range of wavelengths has shown the presence of the organic matrices (Dauphin and Perrin, 1992; Dauphin and Denis, 1999). Amide II bands (near 1550 cm − 1) and amide B band (3060 cm − 1) are sometimes visible in biogenic carbonates. CH2 (present in amino-acids) is near 2510–2520 cm − 1.
Y. Dauphin, A. Denis / Comparati6e Biochemistry and Physiology, Part A 126 (2000) 367–377
2.2.4. Extraction and purification of the organic matrix Shells were immersed in NaClO to remove organic contaminants, rinsed with Milli-Q water, dried and ground into powder. Powdered shells were decalcified in acetic acid at a pH of 4. The entire extract was centrifuged at 21 000× g for 15 min, which separated the supernatant (soluble) and precipitated (insoluble) fractions. The soluble fraction was desalted by exchange with Milli-Q H2O on microconcentrator (Filtron) using a 3 kDa cut off membrane. 2.2.5. UV spectrometry Soluble matrices were dissolved in Milli-Q H2O. UV spectra were recorded on a Shimadzu UV1601 spectrophotometer, equipped with deuterium and tungsten lamps and double beam optics. The monochromator slit aperture is fixed at 2 nm. Before a sample spectra was run, a background spectrum was measured for Milli-Q H2O. All spectra were recorded from 250 to 350 nm. 2.2.6. Chromatographic methods Gel filtration chromatography was used to determine the molecular weights of the soluble fraction. High pressure liquid chromatography (HPLC) separations were done with three TSK columns, equilibrated in Tris – HCl (0.2 M Tris) pH 7.5. TSK G5000PW and G3000PW polymer based and G2000SWXL silica based were chosen according to their range sorting. The materials to be chromatographed were dissolved in the same eluent. The elution patterns were recorded spec-
369
trophotometrically at 226 nm, using a UV2138 detector (Amersham Pharmacia Biotech: APB), and a differential refractometer (APB). The eluent flow was 1 ml/min for polymer based columns and 0.75 ml/min for silica based column. Ion-exchange chromatography was done on Mono-Q (HR5/5, APB) column. Analysis was carried out with eluents A (0.2 M Tris–HCl pH 8.5), and B (eluent A+ 1 M NaCl). An isocratic period of 5 min with eluent A was followed by a linear gradient of eluent A to eluent B in 35 min. The materials to be chromatographed were dissolved in eluent A. The eluent flow was 1 ml/min and the elution patterns were recorded at 226 nm, using a TSP 4100 detector. In this first attempt to compare and estimate the variation of the crossed lamellar layers in mollusc shells, various parameters are described. However, the scheme used to characterize the soluble organic matrices extracted from the shells is not sequential: each analysis has been done on the complete extract.
3. Results
3.1. Microstructures The three Gastropod and Bivalve shells are composed of crossed lamellar layers. This complex structure is composed of lamellae with alternate orientations. According to the orientation of the sections, the observed aspects seem different (Fig. 1).
Fig. 1. Schematic reconstruction of the crossed lamellar layer in mollusca.
370
Y. Dauphin, A. Denis / Comparati6e Biochemistry and Physiology, Part A 126 (2000) 367–377
Fig. 2. (A) Polished and etched section of Cypraea le6iathan showing several layers and various aspects according to the orientation of the first order lamellae. Arrow: growth lines. Formic acid 1%, 20 s ×50. (B) Fracture in the shell of Cypraea le6iathan showing the change in orientation in adjacent layers. Formic acid 1%, 20 s ×170. (C) Polished and etched section of Phalium granulatum with several layers with various orientations of the first order lamellae. Formic acid 5%, 10 s × 280. (D) Detail of structure and second order lamellae in the first order lamellae of Phalium granulatum. Formic acid 5%, 10 s × 705. (E) Polished and etched section of Strombus gigas, showing several layers. Formic acid 5%, 10 s × 50. (F) Polished and etched section of S. gigas showing growth lines (arrows), first and second order lamellae. Formic acid 5%, 10 s × 90. (G) Polished and etched section of Tridacna, showing the inner structure of first order lamellae. Formic acid 5%, 10 s × 103. (H) Polished and etched section of Tridacna, showing third order lamellae. HCl 5%, 8 s ×5400. (I) Fracture of Dosinia ponderosa, with distinct first order lamellae × 955. (J) Fourth order lamellae in Dosinia ponderosa, a granule is underlined (arrow); unetched fracture × 12 000. (K) Polished and etched section of Cardium, showing the inner structure of first order lamellae. Compare with (D). Formic acid 5%, 10 s ×625. (L) Detail of the third order lamellae in Cardium sp. Compare with (H). Formic acid 5%, 10 s × 4250.
Y. Dauphin, A. Denis / Comparati6e Biochemistry and Physiology, Part A 126 (2000) 367–377
371
orientation in adjacent layers is shown in Fig. 2B. The shell of Phalium is also composed with several layers (Fig. 2C), the disposition of which differs from those of Cypraea. A typical aspect of the first order lamellae is visible in Fig. 2D. The arrangement of the layers in the shell of Strombus (Fig. 2E) and the growth lines are shown in Fig. 2F. Two aspects of the structure of the shell of Tridacna are shown in Fig. 2G,H, the second order lamellae with their subdivisions is clearly visible (Fig. 2G), whereas details of the junction and third order lamellae are present in Fig. 2H. The outer layer of Dosinia is a crossed lamellar layer, with distinct first order lamellae (Fig. 2I). In this shell, the fourth order lamellae is visible: the elongated third order lamellae is subdivided into small granules (Fig. 2J). First and second order lamellae of Cardium are typical (Fig. 2K) and the third order lamellae are well developed (Fig. 2L). Each shell is composed of several crossed lamellar layers. The number, the orientations of these sub layers are typical of the taxa, but the basic pattern of first, second and third order lamellar seems similar.
3.2. Bulk composition
Fig. 3. Infra red spectra (4000–450 cm − 1) of the aragonitic crossed lamellar layer of Gastropoda (A) and Bivalvia (B) showing the presence of organic components in the shells. (C) Detail of the amide I (1600–1700 cm − 1) and amide II (1600– 1500 cm − 1) regions showing the presence of amide II band in the gastropod.
The whole section of Cypraea shows that several crossed lamellar layers are arranged with different orientations (Fig. 2A). The change in
All the samples are aragonitic, as shown by FTIR analyses (Fig. 3). The main aragonitic bands are visible: n3 between 1450 and 1490 cm − 1, n1 at 1082 cm − 1, n2 at 863–861 and 844 cm − 1 and n4 at 712–700 cm − 1. Bands of lesser intensity appear at 1785, 1180 and 908 cm − 1 (Dosinia). The wavelength of the n2 doublet (863 cm − 1) is indicative of Sr contents lower than 2000 ppm and Mg contents lower than 400 ppm (Dauphin, 1997). Some organic bands of lesser intensity are also present: amide A band (near 3300 cm − 1), amide I bands (1654 and 1636 cm − 1) and amide II bands (1570–1514 cm − 1). The samples are different in their organic bands: amide A and amide I bands are strong in Strombus (Fig. 3A) and Dosinia (Fig. 3B), and weak in other samples. The number of organic bands is also different: amide I bands are numerous in gastropod shells, whereas they are absent or less abundant in bivalve shells. Two ratios have been calculated from FTIR spectra to estimate the organic/mineral ratio. The first ratio is based on the absorbance intensities of amide A band divided by the 860 cm − 1 part of the n2 band:
372
Y. Dauphin, A. Denis / Comparati6e Biochemistry and Physiology, Part A 126 (2000) 367–377
amide A/860. The second ratio is based on the absorbance intensities of the stronger amide I band divided by the 860 cm − 1 part of the n2 band: amide I/860. In such ratios, all the organic matrices (soluble and insoluble) are counted. According to these ratios (Fig. 4), Dosinia and Strombus contain the highest quantities of organic matrices, Cardium and Tridacna the lowest quantities. It is now accepted that absorbance in the range from 1650 to 1658 cm − 1 is generally associated with the presence of a-helix. Precise interpretation of bands is difficult because there is significant overlap of the a-helical structure with random structures. b-sheet vibrations absorb between 1640 and 1620 cm − 1. Bands centred at 1670 cm − 1 have been assigned to b-turns or turns (Yang et al., 1984; Byler and Susi, 1986; Krimm and Bandekar, 1986). Despite these complications, the FTIR spectra suggest that a major portion of the proteins adopt the in a-helix in Cypraea, Phalium, Strombus and Dosinia. The main structure in Tridacna seems to be turns (1682 cm − 1). b-sheet and random structures are also present. Amide II bands are present only in gastropod shells. According to infrared data, the aragonitic crossed lamellar layers have low Sr and Mg contents. Their organic matrix contents are more variable in quantity and structure than the mineral part.
Fig. 5. Chemical composition of the aragonitic crossed lamellar layers (A,B) and L ratios (C).
3.3. Mineralogy and chemical composition
Fig. 4. Organic/mineral ratios calculated from infrared data, showing the variability of the quantities of the organic matrices (soluble + insoluble) in the studied species.
The aragonitic composition of all the samples is confirmed by EDS analyses. The Na contents are high, while Mg contents are low (Fig. 5). Sr contents are in the range of molluscan aragonite. The substitution ratios Sr2 + and Mg2 + to Ca2 + or L ratios (Loreau, 1982) are also indicative of an aragonitic composition in all the samples (Fig. 5C). They are in accordance with ratios calculated from published data (Milliman, 1974): average L in gastropod shells: 1.491.0, average L in bivalve
Y. Dauphin, A. Denis / Comparati6e Biochemistry and Physiology, Part A 126 (2000) 367–377
shells: 1.591.0 (Loreau, 1982). S contents are low, as they are usually in the organic matrices of aragonitic molluscan layers (Dauphin and Cuif, 1999).
3.4. Soluble organic matrices 3.4.1. Bulk composition The comparison between the UV spectra of a protein (BSA: bovine serum albumin) and the soluble matrices shows that the extracts of the crossed lamellar layers are not pure proteins (Fig. 6). None of the soluble matrices shows a peak near 278 nm, only a shoulder being visible. This shoulder is stronger in Dosinia and Cardium, although the analytical software does not identify a peak. Another difference between the profile of a pure protein and the matrices of the mollusc shells appears between 250 and 280 nm: the BSA spectrum shows a strong increase from 250 to 280 nm, whereas mollusc matrix spectra show a decreasing slope.
373
has large molecular weights (\ 103 kDa), whereas such molecules are absent in Tridacna. Phalium, Dosinia and Strombus show molecular weights :500 kDa according to the elution profiles with TSK G5000. The molecular weights and the composition of the soluble organic matrices extracted from the aragonitic crossed lamellar layers are different in the six studied species. The samples show a large range of non-discrete molecular weights.
3.4.3. Acidity All the samples show a ‘basic’ fraction in the first part of the elution. The acidic parts are variable in intensity and numbers (Fig. 8). Cypraea and Cardium have few acidic parts, whereas these parts are abundant and complex in Dosinia and Tridacna. From this point of view, the soluble organic matrices of the crossed lamellar layers are heterogeneous.
4. Discussion
3.4.2. Molecular weights One set of elution profiles (TSK G3000) is illustrated in Fig. 7. All the profiles are different. At 226 nm, the amounts and the molecular weights differ in each species: the main peak of Tridacna is : 50 kDa, but near 120 kDa in Phalium. Strombus shows two large peaks. The profiles obtained with the refractometer are also different in each shell. Thus, it may be supposed that the sugar contents are unequal. Similar differences have been observed with the TSK G5000PW and G2000SWXL columns (data not shown). TSK G5000 profiles show that Cypraea
4.1. Homogeneity and heterogeneity of the crossed lamellar layers The microstructures and the chemical composition of the crossed lamellar layers of the studied mollusc species are similar. The fourth order lamellae has been previously described in Cardium (Denis, 1972; Uozumi et al., 1972). According to Kobayashi (1994), the third order lamellae are composed of twinned aragonite crystals. All these observations seem to show that the third order lamellae are not monocrystalline. They show
Fig. 6. UV profiles of a protein (BSA) and of the soluble matrices from the crossed lamellar layers, showing that mollusc organic matrices are not pure proteins.
374
Y. Dauphin, A. Denis / Comparati6e Biochemistry and Physiology, Part A 126 (2000) 367–377
Fig. 7. Elution profiles of the soluble matrices obtained by TSK GW3000 column. Each species shows UV and RI profile for the molecular weights.
usual minor element contents for aragonitic mollusc shells (high Na content, low S, Mg and Sr contents). However, the infrared spectra show differences in quality, quantity and structure of the organic matrices. A simple examination with UV spectrometry shows the soluble organic matrices are not pure proteins, and that they are diverse. The molecular weights confirm the variability of the soluble organic matrices, the ratio protein – sugar being different in each species. The molecular weights are not separated in discrete molecules. This result is similar to those obtained with SDS-PAGE electrophoresis in which discrete bands are absent (Weiner et al., 1977). The absence of bands in SDS electrophoresis and isoelectric focussing has also been noticed by Samata (1990). Each sample shows acidic and non-acidic fractions in variable
ratios. Kobayashi (1966) recognized two kinds of organic material by staining decalcified sections of Dosinia: basophilic and eosinophilic. According to non-denaturing isoelectric focussing electrophoresis (IEF), the soluble matrices from Tridacna are acidic, and stained with Coomassie blue and unstained with Alcian blue (Dauphin and Cuif, 1999). Similar results have been obtained with Dosinia and Cypraea matrices (unpublished data). This is in accordance with the low S contents and a high protein/sugar ratio. The above results suggest that the mineral parts of the crossed lamellar layers are similar in the six studied species, whereas the soluble organic matrices are different. However, common characteristics of the soluble organic matrices are also present: they have low S contents, are glycoproteins with high molecular weights and strong
Y. Dauphin, A. Denis / Comparati6e Biochemistry and Physiology, Part A 126 (2000) 367–377
acidity. Gastropoda matrices on the one hand, and Bivalvia matrices on the other hand, do not share obvious common characteristics. It may be suggested that the common features of the organic matrices are responsible for the homogeneity of the microstructures and composition of the mineral parts, whereas the differences are due to taxonomy of the studies samples. According to Carter (1979), the detail microstructures of the crossed lamellar layers are potential ‘useful sources of information for assessing bivalve systematics’, and Carter and Clark (1985) described
375
several shapes of first order crossed lamellae. Unfortunately, very little is known about the microstructures of many species and the organic matrices are not yet studied. Thus, we have not appropriate data to test this hypothesis. A second line of study may be the possible relationship between the environmental conditions and the composition and structure of the crossed lamellar layers. At last, interaction between the physiology and the biochemistry of the shell has not been investigated. It has been shown that amino-acid and monosaccharide contents of zooxanthellate
Fig. 8. Elution profiles of the soluble matrices obtained by MonoQ column. All the samples show acidic and basic parts, with variable portions.
376
Y. Dauphin, A. Denis / Comparati6e Biochemistry and Physiology, Part A 126 (2000) 367–377
and non-zooxanthellate Scleractinia skeletons are significantly different (Cuif et al., 1999). Among molluscs, it is well known that the soft tissues of Tridacna contain zooxanthellate.
4.2. Comparison with other microstructures The organic matrices yielded by the crossed lamellar layers are low relative to nacreous and prismatic layers. The low ‘protein’ content of the crossed lamellar layer of Strombus has been already noted by Nakahara et al. (1981). Infrared data are scarce on mollusc shells (Dauphin and Marin, 1995). However, it seems that the proteins of the nacreous layer of molluscs and the soluble matrix of Strombus exhibit a b-sheet conformation (Weiner, 1984). All the soluble organic matrices extracted from mollusc shells are highly acidic, as shown by their amino acid contents or by exchange ion chromatography (Krampitz et al., 1976; Samata et al., 1980; Samata, 1990). There are strong differences in their S contents, with a correlation with the mineralogy: calcitic layers have higher S contents than the aragonitic layers (Dauphin and Denis, 1995; Dauphin and Cuif, 1999). The soluble matrix of Tridacna is stained with Coomassie blue in non-denaturing IEF gels (Dauphin and Cuif, 1999). Similar stainings were obtained with Sepia shells (Dauphin, 1996), and nacreous layers of Pinna and Pinctada (Marin et al., 1994). There is a paucity of information concerning the amount of sulfated amino acids (cysteine, methionine) due to their degradation during the hydrolysis process. Thus, it is unknown if S is linked with proteins or sugars. Wada (1980) has shown the main role of S for the mineralization process: the crystal formation at the periphery of the inner surface of Pinctada takes place in sulfated organic matrices. Aragonitic skeletons of Scleractinia show high S contents. In non-denaturing IEF, these matrices are unstained with silver or Coomassie blue and stained with Alcian blue. Alcian blue is characteristic of acidic sulfated mucopolysaccharides. Thus it may be supposed that the main part of S is linked with sugars rather than with proteins. The soluble matrices of molluscan shells are yet very poorly known: interaction between the mineral and organic phases is not fully understood, and the example of the nacreous layer is not sufficient. The studied examples show that the range of variation of the organic matrices seems
larger than that of the mineral part of the layers. The mechanisms and rates of the diagenetic changes of the organic matrices are poorly understood. Knowledge of the proteins and sugars contained in carbonate shells will ultimately dictate our understanding of their role in processes of organic degradation and diagenesis, sugars being highly soluble. References Bøggild, O.B., 1930. The shell structure of the molluscs. D. Kgl. Danske Vidensk. Selsk. Skr. (Naturvidensk og mathem.) 9/2 (2), 231 – 326. Byler, D.M., Susi, H., 1986. Examination of the secondary structure of proteins by deconvolved FTIR spectra. Biopolymers 25, 469 – 487. Carter, J.G., 1979. Crossed lamellar signatures: potentially useful sources of information for assessing bivalve systematics. Geol. Soc. Am. 11, 399 Abstract. Carter, J.G., Clark, G.R. II, 1985. Classification and phylogenetic significance of molluscan shell microstructure. In: Broadhead, T.W. (Ed.), MollusksNotes for a Short Course. Studies in Geology, vol. 13. University of Tennessee, pp. 50 – 71. Cuif, J.P., Denis, A., Triclot, M.P., 1985. Ultrastructure de la couche externe du test d’unVeneracea: Dosinia ponderosa (Gray, 1838) (Mollusque, Lamellibranche). Bull. Mus. Natn. Hist. Nat. Sec. 7 (4), 741 – 759. Cuif, J.P., Dauphin, Y., Freiwald, A., Gautret, P., Zibrowius, H., 1999. Biochemical markers of zooxanthellae symbiosis in soluble matrices of skeleton of 24 Scleractinia species. Comp. Biochem. Physiol. 123A (3), 269 – 278. Dauphin, Y., 1996. The organic matrix of coleoid cephalopod shells: molecular weights and isoelectric properties of the soluble matrix in relation to biomineralization processes. Marine Biol. 125, 525 – 529. Dauphin, Y., 1997. Infrared spectra and elemental composition in recent carbonate skeletons: relationships between the n2 band wavelength and Sr and Mg concentrations. Appl. Spectr. 51 (3), 141 – 152. Dauphin, Y., Perrin, C., 1992. Mise en e´vidence de la pre´sence de matie`re organique dans un ciment d’aragonite botryoı¨dale par spectrome´trie infrarouge a` transforme´e de Fourier (FTIR). N. Jb. Geol. Pala¨ont. Abh. 186 (3), 301 – 319. Dauphin, Y., Denis, A., 1995. Incidences d’une analyse statistique de la composition chimique localise´e des coquilles de quelques espe`ces d’Haliotis (Mollusca: Archaeogastropoda). Ann. Sci. Nat. Zool. 16 (13), 7 – 19.
Y. Dauphin, A. Denis / Comparati6e Biochemistry and Physiology, Part A 126 (2000) 367–377
Dauphin, Y., Marin, F., 1995. The compositional analysis of recent cephalopod shell carbohydrates by Fourier transform infrared spectrometry and high performance anion exchange-pulsed amperometric detection. Experientia 51, 278–283. Dauphin, Y., Cuif, J.P., 1999. Relation entre les teneurs en soufre des biomine´raux calcaires et leurs caracte´ristiques mine´ralogiques. Ann. Sci. Nat. 2, 73–85. Dauphin, Y., Denis, A., 1999. Diagene`se compare´e des phases mine´rales et organiques solubles dans les tests aragonitiques de nautiles et d’ammonites. Bull. Soc. Ge´ol. Fr. 170 (3), 355–365. Denis, A., 1972. Essai sur la microstructure du test de Lamellibranches. Trav. Lab. Pale´ont, University of Paris, Orsay, pp. 1–89. Kobayashi, I., 1964. Microscopical observations on the shell structure of Bivalvia — part I Barbatia obtusoides (Nyst). Sci. Rep. Tokyo Kyoiku Daigaku C 82, 295–301. Kobayashi, I., 1966. Submicroscopic observations on the shell structure of Bivalvia — part II Dosinia (Phacosoma) japonica Reeve. Sci. Rep. Sec. C 88, 189–210. Kobayashi, I., 1969. Internal microstructure of the shell of bivalve molluscs. Am. Zool. 9, 663–672. Kobayashi, I., 1973. Data on the differential thermal analyses of bivalvian shells. Bull. Fossil Club Tokyo 7, 1–4. Kobayashi, I., 1994. Twinned aragonite crystals found in the bivalvian crossed lamellar shell structure. J. Geol. Soc. Japan 100 (2), 177–180. Krampitz, G., Engels, J., Cazaux, C., 1976. Biochemical studies on water-soluble proteins and related components of gastropod shells. In: Watabe, N., Wilbur, K.M. (Eds.), The Mechanisms of Mineralization in the Invertebrates and Plants. University of South Carolina Press, Columbia, pp. 155–173. Krimm, S., Bandekar, J., 1986. Vibrational spectroscopy and conformation of peptides, polypeptides, and proteins. In: Anfinsen, C.B., Edsall, J.T., Richards, F.M. (Eds.), Advances in Protein Chemistry, vol. 38. Academic Press, Columbia, pp. 181– 364. Loreau, J.P., 1982. Se´diments aragonitiques et leur gene`se. Me´m. Mus. Natn. Hist. Nat. N.S., Ser. C, Ge´ologie 47, 312. McClintock, C., 1967. Shell structure of patelloid and bellerophontoid gastropods (Mollusca). Bull. Peabody Mus. Nat. Hist. Yale Univ. 22, 1–140. Marin, F., Muyzer, G., Dauphin, Y., 1994. Caracte´risations e´lectrophore´tique et immunologique des matrices organiques solubles des tests de deux Bivalves Pte´riomorphes actuels Pinna nobilis L. et Pinctada margaritifera (L.). C. R. Hebd. Se´anc. Acad. Sc. Paris 318, 1653–1659. .
377
Milliman, J.D., 1974. In: Milliman, J.D., Mu¨ller, G., Fo¨rstner, U. (Eds.), Marine carbonate in recent sedimentary carbonates. Springer Verlag, Berlin, pp. 1 – 375. Nakahara, H., Kakei, M., Bevelander, G., 1981. Studies on the formation of the crossed lamellar structure in the shell of Strombus gigas. Veliger 23 (3), 207 – 211. Samata, T., 1990. Ca-binding glycoproteins in molluscan shells with different types of ultrastructure. Veliger 33 (2), 190 – 201. Samata, T., Sanguansri, P., Cazaux, C., Hamm, M., Engels, J., Krampitz, G., 1980. Biochemical studies on components of mollusc shells. In: Omori, M., Watabe, N. (Eds.), The Mechanisms of Biomineralization in Animals and Plants. Tokai University Press, Tokyo, pp. 37 – 47. Taylor, J.D., Kennedy, W.J., Hall, A., 1969. The shell structure and mineralogy of the Bivalvia. Introduction Nuculacae Trigonacae. Bull. Br. Mus. (Nat. Hist.) Zool. Suppl. 3, 1 – 125. Taylor, J.D., Kennedy, W.J., Hall, A., 1973. The shell structure and mineralogy of the Bivalvia. II — Lucinacea, Clavagellacea, conclusions. Bull. Br. Mus. (Nat. Hist.) Zool. 22 (9), 255 – 294. Uozumi, S., Iwata, K., Togo, Y., 1972. The ultrastructure of the mineral in and the construction of the crossed-lamellar layer in molluscan shell. Contrib. Dept. Geol. Mineral Fac. Sci. Hokkaido Univ. 1236, 447 – 455. Wada, K., 1980. Initiation of mineralization in bivalve molluscs. In: Omori, M., Watabe, N. (Eds.), The Mechanisms of Biomineralization in Animals and Plants. Tokai University Press, Tokyo, pp. 79 – 92. Weiner, S., 1984. Organization of organic matrix components in mineralized tissues. Am. Zool. 24, 945 – 951. Weiner, S., Lowenstam, H.A., Hood, L., 1977. Discrete molecular weight components of the organic matrices of mollusc shells. J. Exp. Marine Biol. Ecol. 30, 45 – 51. White, W.B., 1974. The carbonate minerals. In: Farmer, V.C. (Ed.), The Infrared Spectra of Minerals, Mineralog. Soc. Monogr, vol. 4, pp. 227 – 283. Wilmot, N.V., Barber, D.J., Taylor, J.D., Graham, A.L., 1992. Electron microscopy of molluscan crossed-lamellar microstructure. Phil. Trans. R. Soc. Lond. B 337, 21 – 35. Wise, S.W. Jr, 1971. Shell ultrastructure of the taxodont pelecypod Anadara notabilis (Roding). Eclogae Geol. Helv. 64 (1), 1 – 12. Yang, W.J., Griffiths, P.R., Byler, D.M., Susi, H., 1984. Protein conformation by infrared spectroscopy: resolution enhancement by Fourier self deconvolution. Appl. Spectr. 39 (2), 282 – 287.
www.elsevier.com/locate/cbpa
Structure and composition of the aragonitic crossed lamellar layers in six species of Bivalvia and Gastropoda Y. Dauphin *, A. Denis UMR 8616, Laboratoire de Pale´ontologie, Uni6ersite´ Paris XI, Baˆt. 504, Orsay F-91405, France Received 25 January 2000; received in revised form 3 May 2000; accepted 9 May 2000
Abstract The microstructures, the chemical composition and the soluble organic matrices of the aragonitic crossed lamellar layers of the shells of six species of molluscs have been studied. The microstructures and chemical contents are similar, whereas the quantities of organic matrices are variable. All the soluble matrices are glycoproteins, with low S contents. Their molecular weights, the protein-sugar ratios and acidities are variable. Neither a gastropod nor a bivalve pattern is recognized. The diversity of the organic matrices probably plays a main role in the fossilization processes of mollusc shells. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Biomineralization; Bivalvia; Gastropoda; Crossed lamellar layer; Aragonite; Skeletal organic matrices; DRIFT; HPLC
1. Introduction Shells of the three major mollusc classes: Cephalopoda, Gastropoda and Bivalvia are characteristically layered structures. They are composed of calcium carbonate (aragonite and/or calcite) and organic material. Bøggild (1930) recognized the main microstructural types and the distributions of calcite and aragonite. Among them, the aragonitic nacreous layer has been one of the most intensively studied and provides the basic data to elaborate some models of biomineralization. However, the simple geometric of the nacreous layer and the abundance of its organic matrix do not reflect the complexity of other microstructural types. * Corresponding author. Tel.: + 33-1-69156117; fax: +331-69156123. E-mail address: [email protected] (Y. Dauphin).
The crossed lamellar layer is the most widespread in Gastropoda and Bivalvia. Except Patella, it is aragonitic. The arrangement of aragonitic crystallites was described by Bøggild (1930), then detailed by several authors (Kobayashi 1964, 1966, 1969; McClintock, 1967; Taylor et al., 1969, 1973; Wise 1971; Cuif et al., 1985; Kobayashi, 1994). According to Wilmot et al. (1992), the basic structure of the crossed-lamellar layer is similar in all the studied species. Differential thermal analyses of several Bivalvia shells show that the organic matrix of the crossed lamellar layers differs from those of the prismatic layers (Kobayashi, 1973). Uozumi et al. (1972) described the third order lamellae surrounded by an organic matrix (conchyolin). Weiner et al. (1977) have shown that the protein fraction of the soluble matrices of crossed lamellar layers of some molluscs have variably non-discrete molecular weights. The crossed lamellar layer of Strombus is rich in acidic amino-acids (aspartic and glutamic
1095-6433/00/$ - see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 1 0 9 5 - 6 4 3 3 ( 0 0 ) 0 0 2 1 3 - 0
368
Y. Dauphin, A. Denis / Comparati6e Biochemistry and Physiology, Part A 126 (2000) 367–377
acids) (Nakahara et al., 1981). Samata (1990) confirmed the highly acidic composition of the soluble matrix. More recently, Kobayashi (1994) described a probable organic matrix inside the crystals of the crossed lamellar layer of Glycymeris. However, very little is known about the composition of the organic matrix in these layers. In this paper, we compare the mineral and organic components of the aragonitic crossed lamellar layers in six species of molluscs (three Gastropoda and three Bivalvia).
surements were made using a live time of 100 or 200 s. The electron accelerating voltage applied to the electron gun was 15 kV. The electron beam had a diameter of 100 nm. The elements Na, Mg, Al, P, S, K, Ca, Mn, Fe and Sr were selected to illustrate aspects of shell composition. Several (\ 10) microprobe analyses were made at various locations on each sample. These punctual analyses have been averaged to obtain an ‘individual’ mean. Some elements are below the detection limit of the microprobe (i.e. Mg, Fe). However, they are accounted because their low levels show a good preservation of the shells.
2. Material and methods
2.1. Material Gastropoda: Prosobranchia Cypraea le6iathan Burgess and Arnelle, 1981 (French Polynesia, Tuamotu Islands); Phalium granulatum Born, 1778, unknown origin; Strombus gigas Linne´ 1758 (Caraibes: Martinique, Guadeloupe). Bivalvia: Heterodonta Tridacna sp., unknown origin; Dosinia ponderosa (Gray, 1838) from South California; Cardium sp., unknown origin.
2.2. Methods 2.2.1. Scanning electron microscope (SEM) Fractures and polished etched sections have been observed with Philips 505 and XL30 SEM. Acidic etchings were used to reveal the details of the microstructures of the polished sections. Details of the etchings are given in the figure captions. 2.2.2. Chemical analysis Energy dispersive X-ray microanalysis (EDS) was done using a Philips 505 SEM equipped with a solid state X-ray detector. Quantitative analysis was done on shells by using the Link AN 10 000 ZAF/PB program which estimates peak-to-background ratios. The ZAF/PB method can be applied to bulk specimens with rough surfaces. Small pieces of shells were embedded in an epoxy resin and polished using various grades of diamond paste. A light etching (formic acid, 5%, 15 s) of the polished surfaces was made to reveal the structural details of the samples. The positions of the analysed points with respect to the structural features are, therefore, precisely known. A cobalt sample was used to provide the calibration. Mea-
2.2.3. FTIR Infrared analyses have been carried out on powdered shells. All spectra were recorded at 4 cm − 1 resolution with 64 scans (measurement time \ 4 min) with a string Norton–Beer apodization on a Perkin–Elmer Model 1600 Fourier transform infra-red spectrometer (FTIR), from 4000 to 450 cm − 1. The spectrometer was equipped with a diffuse reflectance accessory, that permits DRIFT measurements with high sensitivity on powders. All spectra were corrected by the Kubelka–Munk function. Before a sample spectra were run, a background spectrum was measured for pure KBr. Sample spectra were automatically ratioed against background to reduce CO2 and H2O bands. They were dried in an oven at 38°C for 1 night. Powdered shells and KBr were mixed (: 5% powdered shell in KBr) and loaded in the sample cup. The mineralogy and composition of shells can be obtained by infrared analysis, even if this method is not usual for such samples. The infrared spectrum of aragonite is characterized by three prominent absorption maxima at 1470–1490 cm − 1 (n3), 858–844 cm − 1 (n2) and 712–700 cm − 1 (n4). Bands of lesser intensity appear at 1785, 1130 and 1060–1080 cm − 1 (n1). From spectra between 2000 and 400 cm − 1, White (1974) concluded that ‘‘The organically derived materials are spectroscopically indistinguishable from inorganic materials’’. However, reexamination of recent and fossil invertebrate shells with a larger range of wavelengths has shown the presence of the organic matrices (Dauphin and Perrin, 1992; Dauphin and Denis, 1999). Amide II bands (near 1550 cm − 1) and amide B band (3060 cm − 1) are sometimes visible in biogenic carbonates. CH2 (present in amino-acids) is near 2510–2520 cm − 1.
Y. Dauphin, A. Denis / Comparati6e Biochemistry and Physiology, Part A 126 (2000) 367–377
2.2.4. Extraction and purification of the organic matrix Shells were immersed in NaClO to remove organic contaminants, rinsed with Milli-Q water, dried and ground into powder. Powdered shells were decalcified in acetic acid at a pH of 4. The entire extract was centrifuged at 21 000× g for 15 min, which separated the supernatant (soluble) and precipitated (insoluble) fractions. The soluble fraction was desalted by exchange with Milli-Q H2O on microconcentrator (Filtron) using a 3 kDa cut off membrane. 2.2.5. UV spectrometry Soluble matrices were dissolved in Milli-Q H2O. UV spectra were recorded on a Shimadzu UV1601 spectrophotometer, equipped with deuterium and tungsten lamps and double beam optics. The monochromator slit aperture is fixed at 2 nm. Before a sample spectra was run, a background spectrum was measured for Milli-Q H2O. All spectra were recorded from 250 to 350 nm. 2.2.6. Chromatographic methods Gel filtration chromatography was used to determine the molecular weights of the soluble fraction. High pressure liquid chromatography (HPLC) separations were done with three TSK columns, equilibrated in Tris – HCl (0.2 M Tris) pH 7.5. TSK G5000PW and G3000PW polymer based and G2000SWXL silica based were chosen according to their range sorting. The materials to be chromatographed were dissolved in the same eluent. The elution patterns were recorded spec-
369
trophotometrically at 226 nm, using a UV2138 detector (Amersham Pharmacia Biotech: APB), and a differential refractometer (APB). The eluent flow was 1 ml/min for polymer based columns and 0.75 ml/min for silica based column. Ion-exchange chromatography was done on Mono-Q (HR5/5, APB) column. Analysis was carried out with eluents A (0.2 M Tris–HCl pH 8.5), and B (eluent A+ 1 M NaCl). An isocratic period of 5 min with eluent A was followed by a linear gradient of eluent A to eluent B in 35 min. The materials to be chromatographed were dissolved in eluent A. The eluent flow was 1 ml/min and the elution patterns were recorded at 226 nm, using a TSP 4100 detector. In this first attempt to compare and estimate the variation of the crossed lamellar layers in mollusc shells, various parameters are described. However, the scheme used to characterize the soluble organic matrices extracted from the shells is not sequential: each analysis has been done on the complete extract.
3. Results
3.1. Microstructures The three Gastropod and Bivalve shells are composed of crossed lamellar layers. This complex structure is composed of lamellae with alternate orientations. According to the orientation of the sections, the observed aspects seem different (Fig. 1).
Fig. 1. Schematic reconstruction of the crossed lamellar layer in mollusca.
370
Y. Dauphin, A. Denis / Comparati6e Biochemistry and Physiology, Part A 126 (2000) 367–377
Fig. 2. (A) Polished and etched section of Cypraea le6iathan showing several layers and various aspects according to the orientation of the first order lamellae. Arrow: growth lines. Formic acid 1%, 20 s ×50. (B) Fracture in the shell of Cypraea le6iathan showing the change in orientation in adjacent layers. Formic acid 1%, 20 s ×170. (C) Polished and etched section of Phalium granulatum with several layers with various orientations of the first order lamellae. Formic acid 5%, 10 s × 280. (D) Detail of structure and second order lamellae in the first order lamellae of Phalium granulatum. Formic acid 5%, 10 s × 705. (E) Polished and etched section of Strombus gigas, showing several layers. Formic acid 5%, 10 s × 50. (F) Polished and etched section of S. gigas showing growth lines (arrows), first and second order lamellae. Formic acid 5%, 10 s × 90. (G) Polished and etched section of Tridacna, showing the inner structure of first order lamellae. Formic acid 5%, 10 s × 103. (H) Polished and etched section of Tridacna, showing third order lamellae. HCl 5%, 8 s ×5400. (I) Fracture of Dosinia ponderosa, with distinct first order lamellae × 955. (J) Fourth order lamellae in Dosinia ponderosa, a granule is underlined (arrow); unetched fracture × 12 000. (K) Polished and etched section of Cardium, showing the inner structure of first order lamellae. Compare with (D). Formic acid 5%, 10 s ×625. (L) Detail of the third order lamellae in Cardium sp. Compare with (H). Formic acid 5%, 10 s × 4250.
Y. Dauphin, A. Denis / Comparati6e Biochemistry and Physiology, Part A 126 (2000) 367–377
371
orientation in adjacent layers is shown in Fig. 2B. The shell of Phalium is also composed with several layers (Fig. 2C), the disposition of which differs from those of Cypraea. A typical aspect of the first order lamellae is visible in Fig. 2D. The arrangement of the layers in the shell of Strombus (Fig. 2E) and the growth lines are shown in Fig. 2F. Two aspects of the structure of the shell of Tridacna are shown in Fig. 2G,H, the second order lamellae with their subdivisions is clearly visible (Fig. 2G), whereas details of the junction and third order lamellae are present in Fig. 2H. The outer layer of Dosinia is a crossed lamellar layer, with distinct first order lamellae (Fig. 2I). In this shell, the fourth order lamellae is visible: the elongated third order lamellae is subdivided into small granules (Fig. 2J). First and second order lamellae of Cardium are typical (Fig. 2K) and the third order lamellae are well developed (Fig. 2L). Each shell is composed of several crossed lamellar layers. The number, the orientations of these sub layers are typical of the taxa, but the basic pattern of first, second and third order lamellar seems similar.
3.2. Bulk composition
Fig. 3. Infra red spectra (4000–450 cm − 1) of the aragonitic crossed lamellar layer of Gastropoda (A) and Bivalvia (B) showing the presence of organic components in the shells. (C) Detail of the amide I (1600–1700 cm − 1) and amide II (1600– 1500 cm − 1) regions showing the presence of amide II band in the gastropod.
The whole section of Cypraea shows that several crossed lamellar layers are arranged with different orientations (Fig. 2A). The change in
All the samples are aragonitic, as shown by FTIR analyses (Fig. 3). The main aragonitic bands are visible: n3 between 1450 and 1490 cm − 1, n1 at 1082 cm − 1, n2 at 863–861 and 844 cm − 1 and n4 at 712–700 cm − 1. Bands of lesser intensity appear at 1785, 1180 and 908 cm − 1 (Dosinia). The wavelength of the n2 doublet (863 cm − 1) is indicative of Sr contents lower than 2000 ppm and Mg contents lower than 400 ppm (Dauphin, 1997). Some organic bands of lesser intensity are also present: amide A band (near 3300 cm − 1), amide I bands (1654 and 1636 cm − 1) and amide II bands (1570–1514 cm − 1). The samples are different in their organic bands: amide A and amide I bands are strong in Strombus (Fig. 3A) and Dosinia (Fig. 3B), and weak in other samples. The number of organic bands is also different: amide I bands are numerous in gastropod shells, whereas they are absent or less abundant in bivalve shells. Two ratios have been calculated from FTIR spectra to estimate the organic/mineral ratio. The first ratio is based on the absorbance intensities of amide A band divided by the 860 cm − 1 part of the n2 band:
372
Y. Dauphin, A. Denis / Comparati6e Biochemistry and Physiology, Part A 126 (2000) 367–377
amide A/860. The second ratio is based on the absorbance intensities of the stronger amide I band divided by the 860 cm − 1 part of the n2 band: amide I/860. In such ratios, all the organic matrices (soluble and insoluble) are counted. According to these ratios (Fig. 4), Dosinia and Strombus contain the highest quantities of organic matrices, Cardium and Tridacna the lowest quantities. It is now accepted that absorbance in the range from 1650 to 1658 cm − 1 is generally associated with the presence of a-helix. Precise interpretation of bands is difficult because there is significant overlap of the a-helical structure with random structures. b-sheet vibrations absorb between 1640 and 1620 cm − 1. Bands centred at 1670 cm − 1 have been assigned to b-turns or turns (Yang et al., 1984; Byler and Susi, 1986; Krimm and Bandekar, 1986). Despite these complications, the FTIR spectra suggest that a major portion of the proteins adopt the in a-helix in Cypraea, Phalium, Strombus and Dosinia. The main structure in Tridacna seems to be turns (1682 cm − 1). b-sheet and random structures are also present. Amide II bands are present only in gastropod shells. According to infrared data, the aragonitic crossed lamellar layers have low Sr and Mg contents. Their organic matrix contents are more variable in quantity and structure than the mineral part.
Fig. 5. Chemical composition of the aragonitic crossed lamellar layers (A,B) and L ratios (C).
3.3. Mineralogy and chemical composition
Fig. 4. Organic/mineral ratios calculated from infrared data, showing the variability of the quantities of the organic matrices (soluble + insoluble) in the studied species.
The aragonitic composition of all the samples is confirmed by EDS analyses. The Na contents are high, while Mg contents are low (Fig. 5). Sr contents are in the range of molluscan aragonite. The substitution ratios Sr2 + and Mg2 + to Ca2 + or L ratios (Loreau, 1982) are also indicative of an aragonitic composition in all the samples (Fig. 5C). They are in accordance with ratios calculated from published data (Milliman, 1974): average L in gastropod shells: 1.491.0, average L in bivalve
Y. Dauphin, A. Denis / Comparati6e Biochemistry and Physiology, Part A 126 (2000) 367–377
shells: 1.591.0 (Loreau, 1982). S contents are low, as they are usually in the organic matrices of aragonitic molluscan layers (Dauphin and Cuif, 1999).
3.4. Soluble organic matrices 3.4.1. Bulk composition The comparison between the UV spectra of a protein (BSA: bovine serum albumin) and the soluble matrices shows that the extracts of the crossed lamellar layers are not pure proteins (Fig. 6). None of the soluble matrices shows a peak near 278 nm, only a shoulder being visible. This shoulder is stronger in Dosinia and Cardium, although the analytical software does not identify a peak. Another difference between the profile of a pure protein and the matrices of the mollusc shells appears between 250 and 280 nm: the BSA spectrum shows a strong increase from 250 to 280 nm, whereas mollusc matrix spectra show a decreasing slope.
373
has large molecular weights (\ 103 kDa), whereas such molecules are absent in Tridacna. Phalium, Dosinia and Strombus show molecular weights :500 kDa according to the elution profiles with TSK G5000. The molecular weights and the composition of the soluble organic matrices extracted from the aragonitic crossed lamellar layers are different in the six studied species. The samples show a large range of non-discrete molecular weights.
3.4.3. Acidity All the samples show a ‘basic’ fraction in the first part of the elution. The acidic parts are variable in intensity and numbers (Fig. 8). Cypraea and Cardium have few acidic parts, whereas these parts are abundant and complex in Dosinia and Tridacna. From this point of view, the soluble organic matrices of the crossed lamellar layers are heterogeneous.
4. Discussion
3.4.2. Molecular weights One set of elution profiles (TSK G3000) is illustrated in Fig. 7. All the profiles are different. At 226 nm, the amounts and the molecular weights differ in each species: the main peak of Tridacna is : 50 kDa, but near 120 kDa in Phalium. Strombus shows two large peaks. The profiles obtained with the refractometer are also different in each shell. Thus, it may be supposed that the sugar contents are unequal. Similar differences have been observed with the TSK G5000PW and G2000SWXL columns (data not shown). TSK G5000 profiles show that Cypraea
4.1. Homogeneity and heterogeneity of the crossed lamellar layers The microstructures and the chemical composition of the crossed lamellar layers of the studied mollusc species are similar. The fourth order lamellae has been previously described in Cardium (Denis, 1972; Uozumi et al., 1972). According to Kobayashi (1994), the third order lamellae are composed of twinned aragonite crystals. All these observations seem to show that the third order lamellae are not monocrystalline. They show
Fig. 6. UV profiles of a protein (BSA) and of the soluble matrices from the crossed lamellar layers, showing that mollusc organic matrices are not pure proteins.
374
Y. Dauphin, A. Denis / Comparati6e Biochemistry and Physiology, Part A 126 (2000) 367–377
Fig. 7. Elution profiles of the soluble matrices obtained by TSK GW3000 column. Each species shows UV and RI profile for the molecular weights.
usual minor element contents for aragonitic mollusc shells (high Na content, low S, Mg and Sr contents). However, the infrared spectra show differences in quality, quantity and structure of the organic matrices. A simple examination with UV spectrometry shows the soluble organic matrices are not pure proteins, and that they are diverse. The molecular weights confirm the variability of the soluble organic matrices, the ratio protein – sugar being different in each species. The molecular weights are not separated in discrete molecules. This result is similar to those obtained with SDS-PAGE electrophoresis in which discrete bands are absent (Weiner et al., 1977). The absence of bands in SDS electrophoresis and isoelectric focussing has also been noticed by Samata (1990). Each sample shows acidic and non-acidic fractions in variable
ratios. Kobayashi (1966) recognized two kinds of organic material by staining decalcified sections of Dosinia: basophilic and eosinophilic. According to non-denaturing isoelectric focussing electrophoresis (IEF), the soluble matrices from Tridacna are acidic, and stained with Coomassie blue and unstained with Alcian blue (Dauphin and Cuif, 1999). Similar results have been obtained with Dosinia and Cypraea matrices (unpublished data). This is in accordance with the low S contents and a high protein/sugar ratio. The above results suggest that the mineral parts of the crossed lamellar layers are similar in the six studied species, whereas the soluble organic matrices are different. However, common characteristics of the soluble organic matrices are also present: they have low S contents, are glycoproteins with high molecular weights and strong
Y. Dauphin, A. Denis / Comparati6e Biochemistry and Physiology, Part A 126 (2000) 367–377
acidity. Gastropoda matrices on the one hand, and Bivalvia matrices on the other hand, do not share obvious common characteristics. It may be suggested that the common features of the organic matrices are responsible for the homogeneity of the microstructures and composition of the mineral parts, whereas the differences are due to taxonomy of the studies samples. According to Carter (1979), the detail microstructures of the crossed lamellar layers are potential ‘useful sources of information for assessing bivalve systematics’, and Carter and Clark (1985) described
375
several shapes of first order crossed lamellae. Unfortunately, very little is known about the microstructures of many species and the organic matrices are not yet studied. Thus, we have not appropriate data to test this hypothesis. A second line of study may be the possible relationship between the environmental conditions and the composition and structure of the crossed lamellar layers. At last, interaction between the physiology and the biochemistry of the shell has not been investigated. It has been shown that amino-acid and monosaccharide contents of zooxanthellate
Fig. 8. Elution profiles of the soluble matrices obtained by MonoQ column. All the samples show acidic and basic parts, with variable portions.
376
Y. Dauphin, A. Denis / Comparati6e Biochemistry and Physiology, Part A 126 (2000) 367–377
and non-zooxanthellate Scleractinia skeletons are significantly different (Cuif et al., 1999). Among molluscs, it is well known that the soft tissues of Tridacna contain zooxanthellate.
4.2. Comparison with other microstructures The organic matrices yielded by the crossed lamellar layers are low relative to nacreous and prismatic layers. The low ‘protein’ content of the crossed lamellar layer of Strombus has been already noted by Nakahara et al. (1981). Infrared data are scarce on mollusc shells (Dauphin and Marin, 1995). However, it seems that the proteins of the nacreous layer of molluscs and the soluble matrix of Strombus exhibit a b-sheet conformation (Weiner, 1984). All the soluble organic matrices extracted from mollusc shells are highly acidic, as shown by their amino acid contents or by exchange ion chromatography (Krampitz et al., 1976; Samata et al., 1980; Samata, 1990). There are strong differences in their S contents, with a correlation with the mineralogy: calcitic layers have higher S contents than the aragonitic layers (Dauphin and Denis, 1995; Dauphin and Cuif, 1999). The soluble matrix of Tridacna is stained with Coomassie blue in non-denaturing IEF gels (Dauphin and Cuif, 1999). Similar stainings were obtained with Sepia shells (Dauphin, 1996), and nacreous layers of Pinna and Pinctada (Marin et al., 1994). There is a paucity of information concerning the amount of sulfated amino acids (cysteine, methionine) due to their degradation during the hydrolysis process. Thus, it is unknown if S is linked with proteins or sugars. Wada (1980) has shown the main role of S for the mineralization process: the crystal formation at the periphery of the inner surface of Pinctada takes place in sulfated organic matrices. Aragonitic skeletons of Scleractinia show high S contents. In non-denaturing IEF, these matrices are unstained with silver or Coomassie blue and stained with Alcian blue. Alcian blue is characteristic of acidic sulfated mucopolysaccharides. Thus it may be supposed that the main part of S is linked with sugars rather than with proteins. The soluble matrices of molluscan shells are yet very poorly known: interaction between the mineral and organic phases is not fully understood, and the example of the nacreous layer is not sufficient. The studied examples show that the range of variation of the organic matrices seems
larger than that of the mineral part of the layers. The mechanisms and rates of the diagenetic changes of the organic matrices are poorly understood. Knowledge of the proteins and sugars contained in carbonate shells will ultimately dictate our understanding of their role in processes of organic degradation and diagenesis, sugars being highly soluble. References Bøggild, O.B., 1930. The shell structure of the molluscs. D. Kgl. Danske Vidensk. Selsk. Skr. (Naturvidensk og mathem.) 9/2 (2), 231 – 326. Byler, D.M., Susi, H., 1986. Examination of the secondary structure of proteins by deconvolved FTIR spectra. Biopolymers 25, 469 – 487. Carter, J.G., 1979. Crossed lamellar signatures: potentially useful sources of information for assessing bivalve systematics. Geol. Soc. Am. 11, 399 Abstract. Carter, J.G., Clark, G.R. II, 1985. Classification and phylogenetic significance of molluscan shell microstructure. In: Broadhead, T.W. (Ed.), MollusksNotes for a Short Course. Studies in Geology, vol. 13. University of Tennessee, pp. 50 – 71. Cuif, J.P., Denis, A., Triclot, M.P., 1985. Ultrastructure de la couche externe du test d’unVeneracea: Dosinia ponderosa (Gray, 1838) (Mollusque, Lamellibranche). Bull. Mus. Natn. Hist. Nat. Sec. 7 (4), 741 – 759. Cuif, J.P., Dauphin, Y., Freiwald, A., Gautret, P., Zibrowius, H., 1999. Biochemical markers of zooxanthellae symbiosis in soluble matrices of skeleton of 24 Scleractinia species. Comp. Biochem. Physiol. 123A (3), 269 – 278. Dauphin, Y., 1996. The organic matrix of coleoid cephalopod shells: molecular weights and isoelectric properties of the soluble matrix in relation to biomineralization processes. Marine Biol. 125, 525 – 529. Dauphin, Y., 1997. Infrared spectra and elemental composition in recent carbonate skeletons: relationships between the n2 band wavelength and Sr and Mg concentrations. Appl. Spectr. 51 (3), 141 – 152. Dauphin, Y., Perrin, C., 1992. Mise en e´vidence de la pre´sence de matie`re organique dans un ciment d’aragonite botryoı¨dale par spectrome´trie infrarouge a` transforme´e de Fourier (FTIR). N. Jb. Geol. Pala¨ont. Abh. 186 (3), 301 – 319. Dauphin, Y., Denis, A., 1995. Incidences d’une analyse statistique de la composition chimique localise´e des coquilles de quelques espe`ces d’Haliotis (Mollusca: Archaeogastropoda). Ann. Sci. Nat. Zool. 16 (13), 7 – 19.
Y. Dauphin, A. Denis / Comparati6e Biochemistry and Physiology, Part A 126 (2000) 367–377
Dauphin, Y., Marin, F., 1995. The compositional analysis of recent cephalopod shell carbohydrates by Fourier transform infrared spectrometry and high performance anion exchange-pulsed amperometric detection. Experientia 51, 278–283. Dauphin, Y., Cuif, J.P., 1999. Relation entre les teneurs en soufre des biomine´raux calcaires et leurs caracte´ristiques mine´ralogiques. Ann. Sci. Nat. 2, 73–85. Dauphin, Y., Denis, A., 1999. Diagene`se compare´e des phases mine´rales et organiques solubles dans les tests aragonitiques de nautiles et d’ammonites. Bull. Soc. Ge´ol. Fr. 170 (3), 355–365. Denis, A., 1972. Essai sur la microstructure du test de Lamellibranches. Trav. Lab. Pale´ont, University of Paris, Orsay, pp. 1–89. Kobayashi, I., 1964. Microscopical observations on the shell structure of Bivalvia — part I Barbatia obtusoides (Nyst). Sci. Rep. Tokyo Kyoiku Daigaku C 82, 295–301. Kobayashi, I., 1966. Submicroscopic observations on the shell structure of Bivalvia — part II Dosinia (Phacosoma) japonica Reeve. Sci. Rep. Sec. C 88, 189–210. Kobayashi, I., 1969. Internal microstructure of the shell of bivalve molluscs. Am. Zool. 9, 663–672. Kobayashi, I., 1973. Data on the differential thermal analyses of bivalvian shells. Bull. Fossil Club Tokyo 7, 1–4. Kobayashi, I., 1994. Twinned aragonite crystals found in the bivalvian crossed lamellar shell structure. J. Geol. Soc. Japan 100 (2), 177–180. Krampitz, G., Engels, J., Cazaux, C., 1976. Biochemical studies on water-soluble proteins and related components of gastropod shells. In: Watabe, N., Wilbur, K.M. (Eds.), The Mechanisms of Mineralization in the Invertebrates and Plants. University of South Carolina Press, Columbia, pp. 155–173. Krimm, S., Bandekar, J., 1986. Vibrational spectroscopy and conformation of peptides, polypeptides, and proteins. In: Anfinsen, C.B., Edsall, J.T., Richards, F.M. (Eds.), Advances in Protein Chemistry, vol. 38. Academic Press, Columbia, pp. 181– 364. Loreau, J.P., 1982. Se´diments aragonitiques et leur gene`se. Me´m. Mus. Natn. Hist. Nat. N.S., Ser. C, Ge´ologie 47, 312. McClintock, C., 1967. Shell structure of patelloid and bellerophontoid gastropods (Mollusca). Bull. Peabody Mus. Nat. Hist. Yale Univ. 22, 1–140. Marin, F., Muyzer, G., Dauphin, Y., 1994. Caracte´risations e´lectrophore´tique et immunologique des matrices organiques solubles des tests de deux Bivalves Pte´riomorphes actuels Pinna nobilis L. et Pinctada margaritifera (L.). C. R. Hebd. Se´anc. Acad. Sc. Paris 318, 1653–1659. .
377
Milliman, J.D., 1974. In: Milliman, J.D., Mu¨ller, G., Fo¨rstner, U. (Eds.), Marine carbonate in recent sedimentary carbonates. Springer Verlag, Berlin, pp. 1 – 375. Nakahara, H., Kakei, M., Bevelander, G., 1981. Studies on the formation of the crossed lamellar structure in the shell of Strombus gigas. Veliger 23 (3), 207 – 211. Samata, T., 1990. Ca-binding glycoproteins in molluscan shells with different types of ultrastructure. Veliger 33 (2), 190 – 201. Samata, T., Sanguansri, P., Cazaux, C., Hamm, M., Engels, J., Krampitz, G., 1980. Biochemical studies on components of mollusc shells. In: Omori, M., Watabe, N. (Eds.), The Mechanisms of Biomineralization in Animals and Plants. Tokai University Press, Tokyo, pp. 37 – 47. Taylor, J.D., Kennedy, W.J., Hall, A., 1969. The shell structure and mineralogy of the Bivalvia. Introduction Nuculacae Trigonacae. Bull. Br. Mus. (Nat. Hist.) Zool. Suppl. 3, 1 – 125. Taylor, J.D., Kennedy, W.J., Hall, A., 1973. The shell structure and mineralogy of the Bivalvia. II — Lucinacea, Clavagellacea, conclusions. Bull. Br. Mus. (Nat. Hist.) Zool. 22 (9), 255 – 294. Uozumi, S., Iwata, K., Togo, Y., 1972. The ultrastructure of the mineral in and the construction of the crossed-lamellar layer in molluscan shell. Contrib. Dept. Geol. Mineral Fac. Sci. Hokkaido Univ. 1236, 447 – 455. Wada, K., 1980. Initiation of mineralization in bivalve molluscs. In: Omori, M., Watabe, N. (Eds.), The Mechanisms of Biomineralization in Animals and Plants. Tokai University Press, Tokyo, pp. 79 – 92. Weiner, S., 1984. Organization of organic matrix components in mineralized tissues. Am. Zool. 24, 945 – 951. Weiner, S., Lowenstam, H.A., Hood, L., 1977. Discrete molecular weight components of the organic matrices of mollusc shells. J. Exp. Marine Biol. Ecol. 30, 45 – 51. White, W.B., 1974. The carbonate minerals. In: Farmer, V.C. (Ed.), The Infrared Spectra of Minerals, Mineralog. Soc. Monogr, vol. 4, pp. 227 – 283. Wilmot, N.V., Barber, D.J., Taylor, J.D., Graham, A.L., 1992. Electron microscopy of molluscan crossed-lamellar microstructure. Phil. Trans. R. Soc. Lond. B 337, 21 – 35. Wise, S.W. Jr, 1971. Shell ultrastructure of the taxodont pelecypod Anadara notabilis (Roding). Eclogae Geol. Helv. 64 (1), 1 – 12. Yang, W.J., Griffiths, P.R., Byler, D.M., Susi, H., 1984. Protein conformation by infrared spectroscopy: resolution enhancement by Fourier self deconvolution. Appl. Spectr. 39 (2), 282 – 287.