Ultrastructure and Mineralization of the Shell of Lingula Unguis Linne, (Inarticualte Brachiopod)
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Title Ultrastructure and Mineralization of the Shell of Lingula unguis Linne, (Inarticualte Brachiopod) Author(s) Iwata, Keiji Citation 北海道大学理学部紀要, 20(1), 35-65 Issue Date 1981-03 Doc URL http://hdl.handle.net/2115/36706 Type bulletin (article) File Information 20_1_p35-65.pdf Instructions for use Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP Jour. Fac. Sci., Hokkaido Univ., Ser. IV, vol. 20, no. 1, March, 1981, pp. 35-65. ULTRASTRUCTURE AND MINERALIZATION OF THE SHELL OF Lingula unguis Linne, (INARTICUALTE BRACHIOPOD) by Keiji Iwata (with 6 text-figures, 1 table, and 14 plates) Abstract Ultrastructure of the shell of Lingula unguis Linne, an inarticulate brachiopod, was studied mainly by electron microscopy to investigate mineralization of the shell. Lingula has a chitino-phosphatic shell, which consists of the periostracum, alternations of organic and mineralized layers, and the punctae. The main part of thc periostracum shows a striped pattern almost perpcndicul'H to the shell surf'lcc and covered b>:, a triple-layered membrane. Showing an elaborate reticulated pattern the organic layer is made up of fine chitinous fibrils. The mineralized layer thins out antero-posteriorly and laterally. Not uniformely mineralized, it shows different grading zones of crystallites at different parts of the shell, as distinguished in the following: amorphous particles or minute granular crystallites; acicular crystallites which are arranged subparallel or intersected at a low angle; coalescent acicular crystallites which intersect irregularly. The organic matrix of the mineralized layer consists of fine proteineous fibrils without periodicity. Cytoplasmic projections of the epithelial cells intrude the punctae, and the distal ends of them adjoin the inner wall of the punctae by a net of fine threads. X-ray diffraction analysis revealed that Lingula shell consisted of carbonate fluorapatite which was similar to francolite. Detected from an electron probe analysis were Ca, P, Mg, F, S, and Na. Amino acid analysis of the shell showed a large amount of alanine and glycine, as well as the characteristic presence of hydroxyproline. A histochemical test of specimens for an organic matrix showed a similarity to collagen, but the ultrastructure of it differed from the typical collagen fibrils. Introduction Among recent and fossil invertebrates inarticulate brachiopods possess phosphatic shells except those which have calcium carbonate sheils, namely, obolellids, trimerellaceans, and craniaceans. From a fossil record it follows that phosphatic mineralization of the shells in inarticulate brachiopods has continued since the Cambrian. Although inarticulate brachio pods fluorished particularly in early Paleozoic, many of them declined or disappeared by late Paleozoic Era. Some descendants of Lingulida and Acrotretida (Lingulidae and Discinidae), however, have survived to date since the Ordovician. Lingula is well known as a living fossil in light of its long phylogenetic history, having a wide distribution especially in warm waters. The in faunal habit of lingulids is recorded, as seen from strata of various ages since the Silurian (McKerrow, 1978). Lingula spends a planktonic life during their early ontogeny, its soft body being covered by a chitinous membrane. Subsequently it sinks to the bottom and secretes a chitinophosphatic shell. A description of the shell structure of Lingula was first given by Gratiolet (1860), in which alternations of the corneous (chitinous) and the calcareous layer were observed and short prisms in the latter were described. Alternations were also described by Chapman (1914). Projections of a mantle epithelium extending into the shell were described by Contribution from the Department of Geology and Mineralogy, Faculty of Science, Hokkaido University, No. 1703. 36 K. Iwata Blochmann (1900). It was demonstrated by Williams and Rowell (1965) that the mineralized layer consisted of extremely fine lamellae which were subparallel or slightly inclined to the shell surface, and pointed out that Gratiolet's prisms were probably mistaken for the punctae which were developed in the mineralized layer. Their suggestion was that many of the punctae were cytoplasmic strands rather than caeca which were found in some punctate articulate brachiopods, and also that alternations of the shell layers had to be formed by a secretory alternation of organic and phosphatic materials at any point of the epithelium. Minerals of the shell of Lingula were studied by Klement (1938), Vinogradov (1953), and McConnel (1963); inorganic composition of the shell was reported by Logan and Hunt (1854), Clark and Wheeler (1922), Vinogradov (1953), and McConnel (1963); amino acid composition of the shell was reported by Jope (1965). Despite their efforts, details of the shell structure remain poorly known. Accordingly, this study looked into ultrastructure and mineralization of the shell, by electron microscopy in observation of Lingula unguis specimens and analyzing minerals by X-ray diffraction and electron probe method, as well as analyzing the shell protein, whereby a structural basis was obtained for a comparison among recent and fossil inarticulate brachiopods and other invertebrates. Materials and methods A total of 40 specimens of Lingula unguis Linne (Text-fig. I) ranging from 1.5 to 3.5 cm in shell length were collected during the lowest spring tide (Oshio), September of Text-fig. 1 Morphology of the shell of Lingula unguis: a,b; inner shell sur face treated with hypochlorite solu tion for one hour; c,d; untreated shell surface. 1979, at a pOint 1.5 km off the sea coast ofSumiyoshi ofVto City, Shimabara Bay, Kyushu, South Japan. It has an infaunal habit and burrows 10- 20 cm in the muddy or sandy bottom, making cylindrical, curved, or V-shaped holes. With tough and flexible pedicles it sticks on the base of the holes, making concretions which are cemented by a mucous and sand grains or muds at the distal end of the pedicles. Dead mollusc shells are also used as a ground mass for pedicle fixation. This species dwells in the sediment not in a scattered manner, but rather in a crowded manner. It coexists with some pelecypods such as Meretrix lusoria and Ruditapes philippillarnm, and polychaetes. A filter feeder, it has the stomoch ULTRASTRUCTURE OF LINGULA SHELL 37 abounding in diatoms. It inhabits even in the brackish environment near the mouth of a river in Shimabara Bay, and the fact showing that Lingula has a wide physiological tolerance against low salinity. It endures an agitation of the bottom sediment during a storm (typhoon), and also survives for a time under a condition of drying up during low tides. Lingula unguis was kept alive in an aquarium until experimental work. For a comparison Lingula reevei (Davidson), Oahu Island, Hawaii, was al.so observed. Optical microscopy and scanning electron microscopy A shell was separated from the soft body and embedded in epoxy resins. A thin section was observed under the binocular and polarized microscope. Some sections were demineralized with O.SM EDTA, and stained with hematoxyline, eosine, van Gieson, Azan, Maroly dyes for examination of a histochemical property of organic materials. Polished specimens were observed under the reflective microscope. A block of a shell specimen embedded in epoxy resins was cut by a diamond wheel and polished with alumina powder. After demineralization by EDTA for 10 minutes or removing organic materials with 10% hypochlorite solution for 10- 20 minutes, specimen was coated with gold in an evaporator and observed under the scanning electron microscope (SEM), JEM U3. Fractured and etched specimens and internal surfaces which were untreated or treated with hypochlorite solution were also observed. Some of the polished specimens were coated with carbon, and a line profile analysis of Ca-Ka and P Ka by an electron microprobe analyzer (EPMA), SMU,-SDS, was carried out in a part of the shell (See P1.3, Fig.l) at IS KV and 2S KV. Transmission electron microscopy Without separating a mantle tissue a shell was fIXed with 1% glutaraldehyde in cacodylate buffer at pH 7.4 for one hour at 4°C, and post-fixed with 1% OsO. in the same buffer for one hour. After dehydration with ethanols this sample was embedded in epoxy reSins, and then thin sections were obtained by ultramicrotomy, using a diamond knife. Some specimens were demineralized with O.SM EDTA and embedded in epoxy resins similarly. Prior to observations some sections were stained with uranyl acetate and lead citrate, and others were npt stained. Thin sections were subjected to a test to see if organic materials are digested by chitinase, pronase, and collagenase, using transmission electron microscope (TEM), JEM 120 U. Identification of shell minerals The shell of Lingula was powdered in agate mortar after immersion in 10% hypochlorite solution for one hour to have excess organic materials removed, and then analyzed with an X-ray diffractometer. A shell specimen was heated in an electric furnace at about SOO°C for one hour and a powdered specimen was also analyzed for a comparison with it. Amino acid analysis After removing the periostracum and muscle fibers, thick mineralized layers of the central part of the shell (3.5 cm) were demineralized with EDTA and analyzed with an 38 K. Iwata automatic amino acid analyzer after hydrolysis with 6 N He!, 104°C, for 24 hours under a reduced condition. An unmineralized sheU margin and a dead sheU (2.5 cm) were also analyzed for a comparison. Results Ultrastructure of the periostracum The sheU of Lingula is covered by an organic periostracum (Text-fig.l-c,d), the color of which is green, darkens as the shell grows. Black or brown iron minerals often adhere on the periostracum. It was well stained by eosine and picric acid. It was not digested by pronase, chitinase, and coUagenase. Its thickness varies with the part of the sheU, about lOll in the central part (Pl.l, Fig.I ). At the sheU margin the thickness of the periostracum decreases below one-third (PI.5, Fig.2).