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Monohydrocalcite in calcareous corpuscles of Mesocestoides corti
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Experimental Parasitology 118 (2008) 54–58 www.elsevier.com/locate/yexpr
Monohydrocalcite in calcareous corpuscles of Mesocestoides corti Mario Sen˜orale-Pose a, Cora Chalar a, Yannicke Dauphin b, Pierre Massard c, Philippe Pradel b, Mo´nica Marı´n a,* a
Seccio´n Bioquı´mica, Facultad de Ciencias, Universidad de la Repu´blica, Igua´ 4225, CP 11400, Montevideo, Uruguay b UMR IDES 8148, Geology, Bat. 504, Universite´ Paris XI-Orsay, 91405-Orsay cedex, France c Department of Geology, Bat. 504, Universite´ Paris XI-Orsay, 91405-Orsay cedex, France Received 3 May 2007; received in revised form 14 June 2007; accepted 25 June 2007 Available online 13 July 2007
Abstract Mesocestoides corti (syn. vogae), as many other cestode platyhelminthes, contains abundant mineralized structures called calcareous corpuscles. These concretions may constitute as much as 40% of the dry weight of the organisms, but their function remains poorly understood. In this work, we reviewed the mineral composition of the calcareous corpuscles of M. corti. X-ray diffraction pattern showed that the major mineral component of the corpuscles is a hydrated form of calcium carbonate, monohydrocalcite, also confirmed by infrared spectrometry. The baseline shift of the X-ray diffraction spectra suggested the presence of amorphous calcium carbonate, accordingly to previous reports, and an organic matrix was confirmed by FTIR. Monohydrocalcite is a rare mineral unusually found in biominerals. Although the significance of monohydrocalcite in biominerals has not been determined, the knowledge of corpuscles composition is of relevance to establish their function and for the elucidation of the mechanisms involved in mineralization processes. 2007 Elsevier Inc. All rights reserved. Index Descriptors and Abbreviations: XRD, X-ray diffraction; FTIR, Fourier transform infrared spectrometry; ACC, Amorphous calcium carbonate; Hydrated calcite; Mineralogy; Calcareous corpuscles; Cestodes; Biomineralization
1. Introduction Biomineral concretions have been described in most invertebrate phyla (Lowenstam, 1981). The platyhelminth parasite Mesocestoides corti (class Cestoda, tapeworms) forms, like other cestodes and trematodes, mineral deposits that are called calcareous corpuscles. These concretions, that may constitute as much as 40% of the dry weight of the organisms, show important variation in shape and size, in the range from 5 to 34 lm in diameter and their function remains poorly understood (for a review, see Vargas-Parada and Laclette, 1999). In cestodes, calcareous corpuscles were proposed to form either intracellularly or extracellularly and, the cell type involved, the place of formation and the mechanism of mineral deposition also appeared *
Corresponding author. Fax: +598 2 525 86 17. E-mail address: [email protected] (M. Marı´n).
0014-4894/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2007.06.011
to be diverse (Vargas-Parada and Laclette, 1999; VargasParada et al., 1999; Smith and Richards, 1993). The ultrastructure of mature calcareous corpuscles appears to be formed by concentric lamellae to which granular material is associated (McCullough and Fairweather, 1987; Smith and Richards, 1993). Since the earlier reports – two centuries ago – to date, the chemical composition of calcareous corpuscles from several organisms has been partially characterized by a variety of techniques. The mineral phase is associated with an organic matrix, which mainly contains polysaccharides, lipids and proteins. X-ray diffraction studies on several species of taeniids found to contain calcium as the principal inorganic component, also magnesium, phosphorus and anions, predominantly carbonate and phosphate. The composition of calcareous corpuscles isolated from M. corti was previously reported (Baldwin et al., 1978; Etges and Marinakis, 1991; Kegley et al., 1969); as well, it was shown
M. Sen˜orale-Pose et al. / Experimental Parasitology 118 (2008) 54–58
that their inorganic composition can vary with the environment when parasites are grown in vitro (Baldwin et al., 1978). X-ray diffraction patterns obtained from isolated corpuscles from Echinococcus granulosus have indicated a poorly crystalline material including the calcite crystal phase of calcium carbonate (Smith and Richards, 1993). Also, the same authors reported that X-ray absorption near-edge spectra of corpuscles from E. granulosus closely resemble the spectrum of brushite, a simple hydrated calcium phosphate (CaHPO4, 2 H2O), and suggested its possible advantage for a ready solubilization of phosphates in metabolic processes (Smith and Richards, 1993). Diverse hypotheses have been proposed concerning the function of the calcareous corpuscles: whilst some authors have considered the role of calcareous corpuscles in terms of calcium metabolism and biomineralization processes, others have focused on the possible protective functions as a buffering system and as reservoirs for metabolic requirements (McCullough and Fairweather, 1987). Also, another hypothesis suggests that calcareous corpuscles might act in fixing wastes (Etges and Marinakis, 1991; Hess, 1980). The knowledge of the composition of calcareous corpuscles is of relevance not only for studying mineral deposit but also for understanding the mechanisms involved in calcification processes. With the aim of studying the calcareous corpuscles biology of cestodes, in this work we reviewed the composition of these corpuscles isolated from the tetrathyridium larval stage and found that the major component of these enigmatic structures is monohydrocalcite, a mineral unusually found in biominerals (Addadi, 2003). 2. Materials and methods 2.1. Parasites Tetrathyridia of M. corti were maintained by intraperitoneal passage in CD1 mice. Two to six months post infection, mice were killed by anaesthesia followed by cervical dislocation, and larvae were recovered from the peritoneal cavity. Parasites were extensively washed with PBS. For histological studies, parasites were washed with sterile PBS and fixed in freshly prepared 4% (w/v) paraformaldehyde in PBS for 1 h. The material was then washed several times in PBS, dehydrated through increasing concentrations of ethanol in PBS and embedded in paraffin wax. Tissue sections of 5–7 lm were cut, mounted onto polylysine-coated slides, dried and stored at 4 C. 2.2. Isolation of calcareous corpuscles Six milliliters of sedimented tetrathyridia were treated with 4% SDS and 15 lg/mL Proteinase K in water in a total volume of 10 mL, during 3 h at 37 C, with gentle agitation. Afterward, from lysed parasites a solid material
55
could be obtained by decantation. The sediment (about 0.8 mL) was mainly composed by calcareous corpuscles and some cellular debris, as could be checked under the microscope. This sediment was rinsed twice with 50 mL of water and submitted to a second treatment with 0.1% SDS, 150 lg/mL Proteinase K in water in a total volume of 2 mL, for 1 h at 37 C, without agitation. Detergent and protease excess were eliminated by rinsing 6 times with 2 mL of water. No cellular debris could be observed under the microscope at this point. After a final wash with 2 mL of 20% ethanol in water, a volume of the same fresh ethanol solution was added to the calcareous corpuscles to avoid microorganism growth. 2.3. Light and electronic microscopy The parasite material was examined with a Nikon E800 microscope and digital images were captured using a CoolSNAP-Pro Monochrome digital camera. Gold-coated images were obtained using scanning electron microscopy (SEM) Jeol 5900 LV model of the Facultad de Ciencias, Montevideo, Uruguay. 2.4. X-ray diffraction (XRD) XRD spectra were obtained on a PANalytical’s X’Pert PRO Diffraction system and X’Pert Pro software, equipped with a Cu anode operating at 45 kV and 40 mA (UMR IDES, Universite´ Paris XI-Orsay). Spectra were first measured from 6 to 80 (2h), with a step size of 0.0167, and step time of 100 sec per step; then more detailed spectra were done from 15 to 55, with a step time of 180 sec per step. Intensities were collected by X’celerator RTMS detector and a Ni filter (kKa1 = 0.1540598 nm). Dried powdered samples were mounted on silicon zero-background holders. A programmable divergence slit was used to expose a surface of 10 · 15 mm for the second acquisition. 2.5. Infrared spectrometry (FTIR) All spectra were recorded at 4 cm 1 resolution with 64 scans (measurement time >4 min) with a strong NortonBeer apodization on a Perkin–Elmer Model 1600 Fourier transform infrared spectrometer (FTIR), in the wavenumber range 4000–450 cm 1 (UMR IDES, Universite´ Paris XI-Orsay). The spectrometer was equipped with a Diffuse Reflectance accessory which permits DRIFT measurements with high sensitivity on powders. All spectra were corrected by the Kubelka–Munk function. The system was purged and maintained permanently under nitrogen to reduce atmospheric CO2 and H2O absorption. Before a spectrum was run, the height of the sample cup was adjusted by using the alignment routine provided by Perkin–Elmer (energy equal or higher than 5%) to optimize the signal. A background spectrum was measured for pure KBr. Sample spectra were automatically ratioed against
56
M. Sen˜orale-Pose et al. / Experimental Parasitology 118 (2008) 54–58
background to minimize CO2 and H2O bands. Correlation coefficients between two spectra of the same samples were about 99%. All samples and KBr were reduced to a powder by grinding with an electric mortar for 10 min to obtain homogeneous granulometry. They were oven-dried at 38 C overnight. Powdered samples and KBr were mixed (about 5% powdered samples in KBr) and loaded into the sample cup (3 mm depth). 3. Results Calcareous corpuscles were isolated from tetrathyridial larval stage of M. corti. Larvae were recovered from the peritoneal cavity of mice and the corpuscles were immediately extracted as described in materials and methods for further analyses. As shown in Fig. 1, corpuscles are irregular round granules, variable in shape and size, in the range of 5–15 l in diameter. The mineral composition was analyzed by X-ray diffraction (XRD) and Fourier transform infrared spectrometry (FTIR). 3.1. X-ray diffraction (XRD) Data lines were identified using the Selected Powder Diffraction Data (published by the Joint Committee on Powder Diffraction Standards Philadelphia, 1974). All peaks of relative intensity greater than 10% of the strongest line are attributable to monohydrocalcite (CaCO3, H2O). Then, the XRD diagram corresponds unambiguously to the mono-
hydrocalcite (card 22-147) (Fig. 2). Nevertheless, baselines of fully crystallized minerals are flat, whereas that of the M. corti mineral is not. Thus it may be suggested that part of the corpuscles is composed of amorphous calcium carbonate (ACC). 3.2. Infrared data The salient features of infrared spectra of the calcium carbonate polymorphs and monohydrocalcite are summarized in Table 1. From the data of Jones (1993), non hydrated polymorphs (calcite, aragonite and vaterite) are characterized by a m2 doublet, whereas monohydrocalcite shows a single band at 873 cm 1. The wavelengths of the m4 band in calcite, and m2 band in aragonite, are sensitive to Mg and Sr contents (Dauphin, 1997, 1999). The main monohydrocalcite bands are visible in M. corti corpuscles with a distinct doublet in the m3 region, a 1067 cm 1 band, a m1 band at 873 cm 1 and a m4 band at 700 cm 1 (Fig. 3). However, the spectra show some differences with the reference spectra (Jones, 1993). First, the wavelengths of the m3 doublet are higher in M. corti corpuscles. Then, the shape of the 1067 cm 1 band differs in the reference spectra and M. corti corpuscles. The sharp band in the non biogenic monohydrocalcite is modified in a broad band in M. corti corpuscles. At last, M. corti corpuscles spectra show one or two bands at 1654 cm 1 and 1647 cm 1, usually assigned to organic bands.
Fig. 1. Histological analysis of Mesocestoides corti larval stage. Light microscopy of a longitudinal tetrathyridium section stained with conventional haematoxylin eosin (A and B). In (B) a magnified panel shows two calcareous corpuscles highlighted by asterisks. (C) Scanning electron micrograph of calcareous corpuscles isolated from tetrathyridia.
M. Sen˜orale-Pose et al. / Experimental Parasitology 118 (2008) 54–58
57
4.1. Composition of M. corti corpuscles
Fig. 2. X-ray diffraction (XRD) of aragonite and calcite, and of M. corti corpuscles.
Table 1 Salient features of infrared spectra of the calcium carbonate polymorphs and monohydrocalcite m3 Monohydrocalcite Calcite Aragonite Vaterite
3232
1789 1798 1789
1767
1703
1477 1489
1069 1162 1119
1409 1429
m1 Monohydrocalcite Calcite Aragonite Vaterite
1484
1012 1083 1089
m2 873 877 858 877
1432 m4
765 848 844 850
700 713 713 745
1409
700
Numbers indicate the wavelengths of the main peaks, in cm 1.
XRD spectra show that M. corti corpuscles are mainly composed of a hydrated calcium carbonate, monohydrocalcite (CaCO3, H2O). Some amorphous calcium carbonate (ACC) is also present. Infrared spectra confirm the presence of monohydrocalcite, but add some interesting features. First, the wavelengths of the m3 band of M.corti corpuscles differ from those of non biogenic monohydrocalcite. However, m3 bands in calcite vary from 1407 to 1435 cm 1, and from 1453 to 1489 cm 1 in aragonite (Adler and Kerr, 1962, 1963a,b; Chester and Elderfield, 1967; Van der Marel and Beutelspacher, 1976; White, 1974). It must be added that the sample shows an unusual variability in this region. Two spectra were acquired the same day, and the wavelengths of the m3 doublet were 1490–1424 cm 1 in the first spectra, and 1496–1430 cm 1 in the second spectra. The wavelengths of other bands are stable. Secondly, the shape of the 1067 cm 1 band has two possible origins. Large bands are usually assigned to poorly crystallized minerals (Beniash et al., 1997). Such an explanation is consistent with the presence of ACC detected with XRD. However, FTIR spectra show the presence of organic matrices. Bands around 3200–3300 cm 1 show the presence of H2O, but they are also due to organic matrices in calcitic and aragonitic skeletons of mollusks and corals (Cuif et al., 2004; Dauphin et al., 2003a). In these skeletons, the organic matrices are composed of proteins and sugars (Dauphin, 2001, 2003b). Proteins and sugars share common bands in FTIR spectra, but the 1050–1150 cm 1 region is only known in sugars. So, the presence of organic components containing sugars can explain the shape of the 1067 cm 1. Furthermore, this broad band may be due to the presence of ACC. The amide A band of M. corti is broader than that of non biogenic monohydrocalcite. As for the 1067 cm 1 band, it may be due to a poorly crystallized mineral or ACC, and to the presence of organic components such as proteins and sugars. This region is always flat in non biogenic carbonates, but shows a large band in biogenic carbonates, such as mollusk and coral skeletons (Dauphin et al., 2003a; Dauphin, 2003b; Dauphin and Denis, 2000). As for the shift of the m3 bands, it may be due to lattice disorder (Gunther et al., 2005). 4.2. Occurrence of monohydrocalcite and ACC in organisms
Fig. 3. Infrared spectrometry (FTIR) spectra of M. corti corpuscles showing the organo-mineral composition.
4. Discussion The Ca-carbonate minerals are the most abundant biogenic minerals. Calcite and aragonite are the most widely produced Ca carbonate polymorphs, whereas vaterite and monohydrocalcite are formed by a limited number of organisms (Addadi, 2003).
Amorphous calcium carbonate is particularly unstable and usually crystallizes in uncontrolled environments. Levi-Kalisman (2002) has shown that ACC in three organisms (ascidian, lobster and Ficus) have the same bulk composition: CaCO3 H2O, similar to that of monohydrocalcite. However, the elemental chemical compositions differ. Because ACC in biological samples are always associated with an organic matrix, it is often said that they are ‘‘stabilized’’ by the organic components. However, it must be said that all biogenic Ca-carbonates contain organic matrices. Mg is also supposed to stabilize ACC, but also increase
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M. Sen˜orale-Pose et al. / Experimental Parasitology 118 (2008) 54–58
solubility (Gunther et al., 2005; Loste et al., 2003). Biogenic occurrence of monohydrocalcite was reported in precipitates induced by several cultured bacterial species and in a guinea pig bladder stone (Rivadeneyra et al., 2004, 1998; Skinner et al., 1977). 5. Conclusions In this work we reviewed the composition of corpuscles isolated from M. corti tetrathyridia. Our results showed that they are mainly composed of monohydrocalcite, ACC and organic matrices. Monohydrocalcite – unusually found in biominerals – was not previously described as major component of corpuscles, and appears as an intracellular product in M. corti concretions. Concerning ACC, their role in biomineralization has been largely neglected but recently, ACC was proposed to have an important basic function in calcium carbonate formation processes as a transient precursor phase of calcite and aragonite (Addadi, 2003). The differences in ACC – structure and composition – observed between organisms could be under genetic control (Addadi, 2003), raising the relevance of the elucidation of the mechanisms involved in the biomineralization process. Acknowledgments The authors wish to thank Laura Domı´nguez and Jenny Saldan˜a (Facultad de Quı´mica, Universidad de la Repu´blica, Uruguay) for the donation of mice infected with M. corti tetrathyridia. This work was supported by PEDECIBA (Uruguay). References Addadi, L., 2003. Taking advantage of disorder: amorphous calcium carbonate and its role in biomineralization. Advanced Materials 15, 959–970. Adler, H.H., Kerr, P.F., 1962. Infrared study of aragonite and calcite. American Mineralogist 47, 700–717. Adler, H.H., Kerr, P.F., 1963a. Infrared absorption frequency trends for anhydrous normal carbonates. American Mineralogist 48, 124–137. Adler, H.H., Kerr, P.F., 1963b. Infrared spectra, symmetry and structure relations of some carbonate minerals. American Mineralogist 48, 839–853. Baldwin, J.L., Berntzen, A.K., Brown, B.W., 1978. Mesocestoides corti: cation concentration in calcareous corpuscles of tetrathyridia grown in vitro. Experimental Parasitology 44, 190–196. Beniash, E., Aizenberg, J., Addadi, L., Weiner, S., 1997. Amorphous calcium carbonate transforms into calcite during sea urchin larval spicule growth. Proceedings of the Royal Society 264, 461–465. Chester, R., Elderfield, H., 1967. The application of infra-red absorption spectroscopy to carbonate mineralogy. Sedimentology 9, 5–21. Cuif, J.P., Dauphin, Y., Berthet, P., Jegoudez, J., 2004. Associated water and organic compounds in coral skeletons: quantitative thermogravimetry coupled to infrared absorption spectrometry. Geochemistry Geophysics Geosystems 5, Q11011. Dauphin, Y., 1997. Infrared spectra and elemental composition in recent carbonate skeletons: relationships between the n2 band wavelength and Sr and Mg concentrations. Applied Spectroscopy 51, 253–258. Dauphin, Y., 1999. Infrared spectra and elemental composition in recent biogenic calcites: relationships between the n4 band wavelength and Sr and Mg concentrations. Applied Spectroscopy 53, 184–190.
Dauphin, Y., 2001. Comparative studies of skeletal soluble matrices from some Scleractinian corals and Molluscs. International Journal of Biological Macromolecules 28, 293–304. Dauphin, Y., 2003b. Soluble organic matrices of the calcitic prismatic shell layers of two Pteriomorphid bivalves. Pinna nobilis and Pinctada margaritifera. Journal of Biological Chemistry 278, 15168– 15177. Dauphin, Y., Denis, A., 2000. Structure and composition of the aragonitic crossed lamellar layers in six species of Bivalvia and Gastropoda. Comparative Biochemistry and Physiology. Part A: Molecular & Integrative Physiology 126, 367–377. Dauphin, Y., Guzman, N., Denis, A., Cuif, J.P., Ortlieb, L., 2003a. Microstructure, nanostructure and composition of the shell of Concholepas concholepas (Gastropoda, Muricidae). Aquatic Living Resources 16, 95–103. Etges, F.J., Marinakis, V., 1991. Formation and excretion of calcareous bodies by the metacestode (Tetrathyridium) of Mesocestoides vogae. Journal of Parasitology 77, 595–602. Gunther, C., Becker, A., Wolf, G., Epple, M., 2005. In vitro synthesis and structural characterization of amorphous calcium carbonate. Zeitschrift fur Anorganische und Allgemeine Chemie 631, 2830–2835. Hess, E., 1980. Ultrastructural study of the tetrathyridium of Mesocestoides corti Hoeppli, 1925: tegument and parenchyma. Zeitschrift fur Parasitenkunde 61, 135–159. Jones, G.C., 1993. Infrared transmission spectra of carbonate minerals. Chapman & Hall, London. Kegley, L.M., Brown, B.W., Berntzen, A.K., 1969. Mesocestoides corti: inorganic components in calcareous corpuscles. Experimental Parasitology 25, 85–92. Levi-Kalisman, Y., 2002. Structural differences between biogenic amorphous calcium carbonate phases using X-ray absorption spectroscopy. Advanced Functional Materials 12, 43–48. Loste, E., Wilson, R.M., Seshadri, R., Meldrum, F.C., 2003. The role of magnesium in stabilising amorphous calcium carbonate and controlling calcite morphology. Journal of Crystal Growth 254, 206–218. Lowenstam, H.A., 1981. Minerals formed by organisms. Science 211, 1126–1131. McCullough, J.S., Fairweather, I., 1987. The structure, composition, formation and possible functions of calcareous corpuscles in Trilocularia acanthiaevulgaris Olsson 1867 (Cestoda, Tetraphyllidea). Parasitology Research 74, 175–182. Rivadeneyra, M.A., Delgado, G., Ramos-Cormenzana, A., Delgado, R., 1998. Biomineralization of carbonates by Halomonas eurihalina in solid and liquid media with different salinities: crystal formation sequence. Research in Microbiology 149, 277–287. Rivadeneyra, M.A., Pa´rraga, J., Delgado, R., Ramos-Cormenzana, A., Delgado, G., 2004. Biomineralization of carbonates by Halobacillus trueperi in solid and liquid media with different salinities. FEMS Microbiology Ecology 48, 39–46. Skinner, H.C.W., Osbaldiston, G.W., Wilner, A.N., 1977. Monohydrocalcite in a guinea pig bladder stone, a novel occurrence. American Mineralogist 62, 273–277. Smith, S.A., Richards, K.S., 1993. Ultrastructure and microanalyses of the calcareous corpuscles of the protoscoleces of Echinococcus granulosus. Parasitology Research 79, 245–250. Van der Marel, H.W., Beutelspacher, H., 1976. Atlas of infrared spectroscopy of clay minerals and their admixtures. Elsevier Scientific Publishing Company, New York, 396 p. Vargas-Parada, L., Laclette, J.P., 1999. Role of the calcareous corpuscles in cestode physiology: a review. Revista Latinoamericana de Microbiologı´a 41, 303–307. Vargas-Parada, L., Merchant, M.T., Willms, K., Laclette, J.P., 1999. Formation of calcareous corpuscles in the lumen of excretory canals of Taenia solium cysticerci. Parasitology Research 85, 88–92. White, W.B., 1974. The carbonate minerals. In: Farmer (Ed.), The Infrared Spectra of Minerals. Mineralogical Society, Monograph 4, London, pp. 227–284.
Experimental Parasitology 118 (2008) 54–58 www.elsevier.com/locate/yexpr
Monohydrocalcite in calcareous corpuscles of Mesocestoides corti Mario Sen˜orale-Pose a, Cora Chalar a, Yannicke Dauphin b, Pierre Massard c, Philippe Pradel b, Mo´nica Marı´n a,* a
Seccio´n Bioquı´mica, Facultad de Ciencias, Universidad de la Repu´blica, Igua´ 4225, CP 11400, Montevideo, Uruguay b UMR IDES 8148, Geology, Bat. 504, Universite´ Paris XI-Orsay, 91405-Orsay cedex, France c Department of Geology, Bat. 504, Universite´ Paris XI-Orsay, 91405-Orsay cedex, France Received 3 May 2007; received in revised form 14 June 2007; accepted 25 June 2007 Available online 13 July 2007
Abstract Mesocestoides corti (syn. vogae), as many other cestode platyhelminthes, contains abundant mineralized structures called calcareous corpuscles. These concretions may constitute as much as 40% of the dry weight of the organisms, but their function remains poorly understood. In this work, we reviewed the mineral composition of the calcareous corpuscles of M. corti. X-ray diffraction pattern showed that the major mineral component of the corpuscles is a hydrated form of calcium carbonate, monohydrocalcite, also confirmed by infrared spectrometry. The baseline shift of the X-ray diffraction spectra suggested the presence of amorphous calcium carbonate, accordingly to previous reports, and an organic matrix was confirmed by FTIR. Monohydrocalcite is a rare mineral unusually found in biominerals. Although the significance of monohydrocalcite in biominerals has not been determined, the knowledge of corpuscles composition is of relevance to establish their function and for the elucidation of the mechanisms involved in mineralization processes. 2007 Elsevier Inc. All rights reserved. Index Descriptors and Abbreviations: XRD, X-ray diffraction; FTIR, Fourier transform infrared spectrometry; ACC, Amorphous calcium carbonate; Hydrated calcite; Mineralogy; Calcareous corpuscles; Cestodes; Biomineralization
1. Introduction Biomineral concretions have been described in most invertebrate phyla (Lowenstam, 1981). The platyhelminth parasite Mesocestoides corti (class Cestoda, tapeworms) forms, like other cestodes and trematodes, mineral deposits that are called calcareous corpuscles. These concretions, that may constitute as much as 40% of the dry weight of the organisms, show important variation in shape and size, in the range from 5 to 34 lm in diameter and their function remains poorly understood (for a review, see Vargas-Parada and Laclette, 1999). In cestodes, calcareous corpuscles were proposed to form either intracellularly or extracellularly and, the cell type involved, the place of formation and the mechanism of mineral deposition also appeared *
Corresponding author. Fax: +598 2 525 86 17. E-mail address: [email protected] (M. Marı´n).
0014-4894/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2007.06.011
to be diverse (Vargas-Parada and Laclette, 1999; VargasParada et al., 1999; Smith and Richards, 1993). The ultrastructure of mature calcareous corpuscles appears to be formed by concentric lamellae to which granular material is associated (McCullough and Fairweather, 1987; Smith and Richards, 1993). Since the earlier reports – two centuries ago – to date, the chemical composition of calcareous corpuscles from several organisms has been partially characterized by a variety of techniques. The mineral phase is associated with an organic matrix, which mainly contains polysaccharides, lipids and proteins. X-ray diffraction studies on several species of taeniids found to contain calcium as the principal inorganic component, also magnesium, phosphorus and anions, predominantly carbonate and phosphate. The composition of calcareous corpuscles isolated from M. corti was previously reported (Baldwin et al., 1978; Etges and Marinakis, 1991; Kegley et al., 1969); as well, it was shown
M. Sen˜orale-Pose et al. / Experimental Parasitology 118 (2008) 54–58
that their inorganic composition can vary with the environment when parasites are grown in vitro (Baldwin et al., 1978). X-ray diffraction patterns obtained from isolated corpuscles from Echinococcus granulosus have indicated a poorly crystalline material including the calcite crystal phase of calcium carbonate (Smith and Richards, 1993). Also, the same authors reported that X-ray absorption near-edge spectra of corpuscles from E. granulosus closely resemble the spectrum of brushite, a simple hydrated calcium phosphate (CaHPO4, 2 H2O), and suggested its possible advantage for a ready solubilization of phosphates in metabolic processes (Smith and Richards, 1993). Diverse hypotheses have been proposed concerning the function of the calcareous corpuscles: whilst some authors have considered the role of calcareous corpuscles in terms of calcium metabolism and biomineralization processes, others have focused on the possible protective functions as a buffering system and as reservoirs for metabolic requirements (McCullough and Fairweather, 1987). Also, another hypothesis suggests that calcareous corpuscles might act in fixing wastes (Etges and Marinakis, 1991; Hess, 1980). The knowledge of the composition of calcareous corpuscles is of relevance not only for studying mineral deposit but also for understanding the mechanisms involved in calcification processes. With the aim of studying the calcareous corpuscles biology of cestodes, in this work we reviewed the composition of these corpuscles isolated from the tetrathyridium larval stage and found that the major component of these enigmatic structures is monohydrocalcite, a mineral unusually found in biominerals (Addadi, 2003). 2. Materials and methods 2.1. Parasites Tetrathyridia of M. corti were maintained by intraperitoneal passage in CD1 mice. Two to six months post infection, mice were killed by anaesthesia followed by cervical dislocation, and larvae were recovered from the peritoneal cavity. Parasites were extensively washed with PBS. For histological studies, parasites were washed with sterile PBS and fixed in freshly prepared 4% (w/v) paraformaldehyde in PBS for 1 h. The material was then washed several times in PBS, dehydrated through increasing concentrations of ethanol in PBS and embedded in paraffin wax. Tissue sections of 5–7 lm were cut, mounted onto polylysine-coated slides, dried and stored at 4 C. 2.2. Isolation of calcareous corpuscles Six milliliters of sedimented tetrathyridia were treated with 4% SDS and 15 lg/mL Proteinase K in water in a total volume of 10 mL, during 3 h at 37 C, with gentle agitation. Afterward, from lysed parasites a solid material
55
could be obtained by decantation. The sediment (about 0.8 mL) was mainly composed by calcareous corpuscles and some cellular debris, as could be checked under the microscope. This sediment was rinsed twice with 50 mL of water and submitted to a second treatment with 0.1% SDS, 150 lg/mL Proteinase K in water in a total volume of 2 mL, for 1 h at 37 C, without agitation. Detergent and protease excess were eliminated by rinsing 6 times with 2 mL of water. No cellular debris could be observed under the microscope at this point. After a final wash with 2 mL of 20% ethanol in water, a volume of the same fresh ethanol solution was added to the calcareous corpuscles to avoid microorganism growth. 2.3. Light and electronic microscopy The parasite material was examined with a Nikon E800 microscope and digital images were captured using a CoolSNAP-Pro Monochrome digital camera. Gold-coated images were obtained using scanning electron microscopy (SEM) Jeol 5900 LV model of the Facultad de Ciencias, Montevideo, Uruguay. 2.4. X-ray diffraction (XRD) XRD spectra were obtained on a PANalytical’s X’Pert PRO Diffraction system and X’Pert Pro software, equipped with a Cu anode operating at 45 kV and 40 mA (UMR IDES, Universite´ Paris XI-Orsay). Spectra were first measured from 6 to 80 (2h), with a step size of 0.0167, and step time of 100 sec per step; then more detailed spectra were done from 15 to 55, with a step time of 180 sec per step. Intensities were collected by X’celerator RTMS detector and a Ni filter (kKa1 = 0.1540598 nm). Dried powdered samples were mounted on silicon zero-background holders. A programmable divergence slit was used to expose a surface of 10 · 15 mm for the second acquisition. 2.5. Infrared spectrometry (FTIR) All spectra were recorded at 4 cm 1 resolution with 64 scans (measurement time >4 min) with a strong NortonBeer apodization on a Perkin–Elmer Model 1600 Fourier transform infrared spectrometer (FTIR), in the wavenumber range 4000–450 cm 1 (UMR IDES, Universite´ Paris XI-Orsay). The spectrometer was equipped with a Diffuse Reflectance accessory which permits DRIFT measurements with high sensitivity on powders. All spectra were corrected by the Kubelka–Munk function. The system was purged and maintained permanently under nitrogen to reduce atmospheric CO2 and H2O absorption. Before a spectrum was run, the height of the sample cup was adjusted by using the alignment routine provided by Perkin–Elmer (energy equal or higher than 5%) to optimize the signal. A background spectrum was measured for pure KBr. Sample spectra were automatically ratioed against
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background to minimize CO2 and H2O bands. Correlation coefficients between two spectra of the same samples were about 99%. All samples and KBr were reduced to a powder by grinding with an electric mortar for 10 min to obtain homogeneous granulometry. They were oven-dried at 38 C overnight. Powdered samples and KBr were mixed (about 5% powdered samples in KBr) and loaded into the sample cup (3 mm depth). 3. Results Calcareous corpuscles were isolated from tetrathyridial larval stage of M. corti. Larvae were recovered from the peritoneal cavity of mice and the corpuscles were immediately extracted as described in materials and methods for further analyses. As shown in Fig. 1, corpuscles are irregular round granules, variable in shape and size, in the range of 5–15 l in diameter. The mineral composition was analyzed by X-ray diffraction (XRD) and Fourier transform infrared spectrometry (FTIR). 3.1. X-ray diffraction (XRD) Data lines were identified using the Selected Powder Diffraction Data (published by the Joint Committee on Powder Diffraction Standards Philadelphia, 1974). All peaks of relative intensity greater than 10% of the strongest line are attributable to monohydrocalcite (CaCO3, H2O). Then, the XRD diagram corresponds unambiguously to the mono-
hydrocalcite (card 22-147) (Fig. 2). Nevertheless, baselines of fully crystallized minerals are flat, whereas that of the M. corti mineral is not. Thus it may be suggested that part of the corpuscles is composed of amorphous calcium carbonate (ACC). 3.2. Infrared data The salient features of infrared spectra of the calcium carbonate polymorphs and monohydrocalcite are summarized in Table 1. From the data of Jones (1993), non hydrated polymorphs (calcite, aragonite and vaterite) are characterized by a m2 doublet, whereas monohydrocalcite shows a single band at 873 cm 1. The wavelengths of the m4 band in calcite, and m2 band in aragonite, are sensitive to Mg and Sr contents (Dauphin, 1997, 1999). The main monohydrocalcite bands are visible in M. corti corpuscles with a distinct doublet in the m3 region, a 1067 cm 1 band, a m1 band at 873 cm 1 and a m4 band at 700 cm 1 (Fig. 3). However, the spectra show some differences with the reference spectra (Jones, 1993). First, the wavelengths of the m3 doublet are higher in M. corti corpuscles. Then, the shape of the 1067 cm 1 band differs in the reference spectra and M. corti corpuscles. The sharp band in the non biogenic monohydrocalcite is modified in a broad band in M. corti corpuscles. At last, M. corti corpuscles spectra show one or two bands at 1654 cm 1 and 1647 cm 1, usually assigned to organic bands.
Fig. 1. Histological analysis of Mesocestoides corti larval stage. Light microscopy of a longitudinal tetrathyridium section stained with conventional haematoxylin eosin (A and B). In (B) a magnified panel shows two calcareous corpuscles highlighted by asterisks. (C) Scanning electron micrograph of calcareous corpuscles isolated from tetrathyridia.
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4.1. Composition of M. corti corpuscles
Fig. 2. X-ray diffraction (XRD) of aragonite and calcite, and of M. corti corpuscles.
Table 1 Salient features of infrared spectra of the calcium carbonate polymorphs and monohydrocalcite m3 Monohydrocalcite Calcite Aragonite Vaterite
3232
1789 1798 1789
1767
1703
1477 1489
1069 1162 1119
1409 1429
m1 Monohydrocalcite Calcite Aragonite Vaterite
1484
1012 1083 1089
m2 873 877 858 877
1432 m4
765 848 844 850
700 713 713 745
1409
700
Numbers indicate the wavelengths of the main peaks, in cm 1.
XRD spectra show that M. corti corpuscles are mainly composed of a hydrated calcium carbonate, monohydrocalcite (CaCO3, H2O). Some amorphous calcium carbonate (ACC) is also present. Infrared spectra confirm the presence of monohydrocalcite, but add some interesting features. First, the wavelengths of the m3 band of M.corti corpuscles differ from those of non biogenic monohydrocalcite. However, m3 bands in calcite vary from 1407 to 1435 cm 1, and from 1453 to 1489 cm 1 in aragonite (Adler and Kerr, 1962, 1963a,b; Chester and Elderfield, 1967; Van der Marel and Beutelspacher, 1976; White, 1974). It must be added that the sample shows an unusual variability in this region. Two spectra were acquired the same day, and the wavelengths of the m3 doublet were 1490–1424 cm 1 in the first spectra, and 1496–1430 cm 1 in the second spectra. The wavelengths of other bands are stable. Secondly, the shape of the 1067 cm 1 band has two possible origins. Large bands are usually assigned to poorly crystallized minerals (Beniash et al., 1997). Such an explanation is consistent with the presence of ACC detected with XRD. However, FTIR spectra show the presence of organic matrices. Bands around 3200–3300 cm 1 show the presence of H2O, but they are also due to organic matrices in calcitic and aragonitic skeletons of mollusks and corals (Cuif et al., 2004; Dauphin et al., 2003a). In these skeletons, the organic matrices are composed of proteins and sugars (Dauphin, 2001, 2003b). Proteins and sugars share common bands in FTIR spectra, but the 1050–1150 cm 1 region is only known in sugars. So, the presence of organic components containing sugars can explain the shape of the 1067 cm 1. Furthermore, this broad band may be due to the presence of ACC. The amide A band of M. corti is broader than that of non biogenic monohydrocalcite. As for the 1067 cm 1 band, it may be due to a poorly crystallized mineral or ACC, and to the presence of organic components such as proteins and sugars. This region is always flat in non biogenic carbonates, but shows a large band in biogenic carbonates, such as mollusk and coral skeletons (Dauphin et al., 2003a; Dauphin, 2003b; Dauphin and Denis, 2000). As for the shift of the m3 bands, it may be due to lattice disorder (Gunther et al., 2005). 4.2. Occurrence of monohydrocalcite and ACC in organisms
Fig. 3. Infrared spectrometry (FTIR) spectra of M. corti corpuscles showing the organo-mineral composition.
4. Discussion The Ca-carbonate minerals are the most abundant biogenic minerals. Calcite and aragonite are the most widely produced Ca carbonate polymorphs, whereas vaterite and monohydrocalcite are formed by a limited number of organisms (Addadi, 2003).
Amorphous calcium carbonate is particularly unstable and usually crystallizes in uncontrolled environments. Levi-Kalisman (2002) has shown that ACC in three organisms (ascidian, lobster and Ficus) have the same bulk composition: CaCO3 H2O, similar to that of monohydrocalcite. However, the elemental chemical compositions differ. Because ACC in biological samples are always associated with an organic matrix, it is often said that they are ‘‘stabilized’’ by the organic components. However, it must be said that all biogenic Ca-carbonates contain organic matrices. Mg is also supposed to stabilize ACC, but also increase
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solubility (Gunther et al., 2005; Loste et al., 2003). Biogenic occurrence of monohydrocalcite was reported in precipitates induced by several cultured bacterial species and in a guinea pig bladder stone (Rivadeneyra et al., 2004, 1998; Skinner et al., 1977). 5. Conclusions In this work we reviewed the composition of corpuscles isolated from M. corti tetrathyridia. Our results showed that they are mainly composed of monohydrocalcite, ACC and organic matrices. Monohydrocalcite – unusually found in biominerals – was not previously described as major component of corpuscles, and appears as an intracellular product in M. corti concretions. Concerning ACC, their role in biomineralization has been largely neglected but recently, ACC was proposed to have an important basic function in calcium carbonate formation processes as a transient precursor phase of calcite and aragonite (Addadi, 2003). The differences in ACC – structure and composition – observed between organisms could be under genetic control (Addadi, 2003), raising the relevance of the elucidation of the mechanisms involved in the biomineralization process. Acknowledgments The authors wish to thank Laura Domı´nguez and Jenny Saldan˜a (Facultad de Quı´mica, Universidad de la Repu´blica, Uruguay) for the donation of mice infected with M. corti tetrathyridia. This work was supported by PEDECIBA (Uruguay). References Addadi, L., 2003. Taking advantage of disorder: amorphous calcium carbonate and its role in biomineralization. Advanced Materials 15, 959–970. Adler, H.H., Kerr, P.F., 1962. Infrared study of aragonite and calcite. American Mineralogist 47, 700–717. Adler, H.H., Kerr, P.F., 1963a. Infrared absorption frequency trends for anhydrous normal carbonates. American Mineralogist 48, 124–137. Adler, H.H., Kerr, P.F., 1963b. Infrared spectra, symmetry and structure relations of some carbonate minerals. American Mineralogist 48, 839–853. Baldwin, J.L., Berntzen, A.K., Brown, B.W., 1978. Mesocestoides corti: cation concentration in calcareous corpuscles of tetrathyridia grown in vitro. Experimental Parasitology 44, 190–196. Beniash, E., Aizenberg, J., Addadi, L., Weiner, S., 1997. Amorphous calcium carbonate transforms into calcite during sea urchin larval spicule growth. Proceedings of the Royal Society 264, 461–465. Chester, R., Elderfield, H., 1967. The application of infra-red absorption spectroscopy to carbonate mineralogy. Sedimentology 9, 5–21. Cuif, J.P., Dauphin, Y., Berthet, P., Jegoudez, J., 2004. Associated water and organic compounds in coral skeletons: quantitative thermogravimetry coupled to infrared absorption spectrometry. Geochemistry Geophysics Geosystems 5, Q11011. Dauphin, Y., 1997. Infrared spectra and elemental composition in recent carbonate skeletons: relationships between the n2 band wavelength and Sr and Mg concentrations. Applied Spectroscopy 51, 253–258. Dauphin, Y., 1999. Infrared spectra and elemental composition in recent biogenic calcites: relationships between the n4 band wavelength and Sr and Mg concentrations. Applied Spectroscopy 53, 184–190.
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