Ultrastructural studies on the brachiopod pedicle SARAH MACKAY AND ROGER A. H E W

LETH AIA Mackay, Sarah & Hewitt, Roger A. 1978 10 15: Ultrastructural studies on the brachiopod pedicle. Lerhaia, Vol. 11, pp. 331-339. Oslo. ISSN 0024-1164.

Studies of the ultrastructure of representative articulate and inarticulate pedicles show that there are similarities in secretory activity between pedicle epithelia of both classes and the outer mantle epithelium of the inarticulates. Rootlet epithelial cells of the articulate pedicle produce small vesicles which pass to the junction between rootlet and substrate and probably play a part in the breakdown of the substrate. Scanning electron microscopy shows that dissolution of a bivalve shell acting as a substrate often follows the boundary of a single nacre tablet, and transmission electron microscopy shows that the rootlet extends its infiltration along the conchiolin walls. The inarticulate pedicle ending is modified to collect adherent sand grains.

Sarah Mackay and Roger A . Hewitt. The Department of Geological Sciences, The University of Birmingham, B15 2TT; 6th February, 1978.

Few studies have been made on the structure of the brachiopod pedicle. Recent papers have been mainly concerned with function: one showed that the pedicle of an inarticulate is not its principal burrowing organ Vhayer & Steele- PetroviC 1975), and another examined the borings produced by articulate pedicles (Bromley & Sur- lyk 1973). There has been no work on the ultra- structure of the pedicle of either class. For these reasons, electron microscopy has been used to study the relationship between form and func- tion in the brachiopod pedicle.

Terebratulina retusa (Linnaeus) has been cho- sen as a representative articulate brachiopod and Glottidia pyramidata (Simpson) as a representa- tive inarticulate. Additional observations were made on the gross structure of pedicles of Lin- gula anatina (Bruguibre) and Laqueus californi- cus (Koch). Fossil pedicle impressions were ex- amined in the shell of Miocene Terebratula tere- bratula (Linnaeus).

Living specimens of Glottidia pyramidata were supplied by the Gulf Specimen Company, Panacea, Florida, and those of Terebratulina retusa were supplied by the University Marine Biological Station, Millport, Isle of Cumbrae, Scotland. The brachiopcds were maintained in aquaria in accordance with the method of McCammon (1975: 15) until required for fixation. Material was prepared for examination in the transmission electron microscope (TEM) by a double fixation method.Fixa- tion in 3% glutaraldehyde made up in 3% sodium chloride solution and buffered to pH 7.2 with phosphate buffer for two hours in the cold was followed by a wash in phosphate buffer. After decalcification in 10% EDTA and a wash in 0.2M sucrose solution the material was post-fixed in 2% osmium tetroxide; all these solutions were buffered to pH 7.2 with phosphate buffer. Dehydration in ethanol was followed by embedding in

Taab resin and sections were stained in aqueous lead citrate and uranyl acetate, and examined in an A.E.I. Corinth TEM. Preparation of material for examination in the scanning elec- tron microscope (SEM) was similar up to the stage of dehydra- tion, which was carried out in acetone and followed by critical point drying. The SEM material was coated with gold/palladi- um under a high vacuum and examined under progressively increasing accelerating voltages (2.5-25 kV) with a Cambridge S2A Stereoscan.

Pedicle structure The terebratulid shell is joined to the substrate by the pedicle and can be rotated by the adjustor muscles within its shell cavity. The form of the pedicle varies with the nature of the substrate. For example, in Chlidonophora it is divided into threadlike branches usually connected with Glo- bigerina ooze (Blochman in Chun lm), while in Terebratulina and Laqueus it is often a short trunk-like structure attached to living or dead shells of bivalves such as Modiolus. All three examples have distal rootlets which can pene- trate and form a bond with the substrate, but the way in which this is achieved has not been stu- died.

The terebratulid pedicle consists of a core of connective tissue, a pedicle epithelium and an external chitinous cuticle (Fig. 1A). The distal end is subdivided into rootlets (Fig. 2A and B). The lingulid pedicle differs as there are no rootlets and there is a central coelomic canal surrounded by a coelomic epithelium and a ring of muscular tissue. Outside the muscular layer is

332 Sarah Mackay and Roger A . Hewitt LETHAL4 11 (1978)

Fig. / . 0 A. Electron micrograph of the Terebratulina pedicle showing the pedicle epithelium (at the lower half of the picture) and the cuticle above with its dense exterior edge. x 3600. 0 B. Electron micrograph of the Glottidia pedicle showing the coelomic epithelium at the lower edge of the picture, with the muscular layer, connective tissue and pedicle epithelium above it. ~ 3 6 0 0 .

a thin layer of connective tissue, then the pedicle epithelium and outer cuticle as in the terebratulid pedicle (Fig. IB).

The coelom of Glottidia is filled with a coelom- ic fluid carrying erythrocytes and amoebocytes and is encircled by a layer of coelomic epitheli- um about 4.6 pm high. The fine structure of the coelomic epithelium is similar in all parts of the Glottidia pedicle; the cells are characterized by a microvillous border and electron-dense droplets

on average 0.76 pm in diameter. Large Lingula specimens show that the coelom expands gradu- ally to form a distal bulb, while the ring of mus- cular tissue is reduced in thickness (Fig. 3B and C). Both Lingula and Glottidia muscle fibres run longitudinally along a spiral axis and are supported by a sheet of connective tissue from 3 to 6 pm thick. Transmission electron microsco- py shows that the sheet is composed of striated collagen fibres which run circularly. Scanning

LETHAL4 11 (1978) Ultrastructure of the brachiopod pedicle 333

Fig. 2 . Scanning electron micrographs of the Terebratulina pedicle. 0 A. The distal end of the pedicle with rootlets. x 82. 0 B. A transverse section through the pedicle trunk and rootlets. x59.

electron microscopy of the connective tissue of Lingula shows grooves about 1 pm deep spaced at intervals of 60 pm along the axis of the pedi- cle, .subordinate longitudinal ridges about 3 pm wide and bundles of collagen fibres about 1 pm in diameter.

The cental connective tissue of the proximal Terebratulina pedicle consist largely of densely packed longitudinal collagen fibres. Fat cells, fibroblasts and areas of transversely running col- lagen fibres are also found. Striated collagen fi- bres are seen in the TEM and finest fibrils (on average 0.1 pm in diameter) seen in the SEM are often grouped into bundles about 0.4 pm in dia- meter.

The cuticle of Lingula specimens has a con- stant thickness of about 0.7 mm in the proximal half of the pedicle and thickens distally. It does, however, form a thick double fold around the rear of the shell and attenuates over the last 10 mm of the distal coelomic bulb (Figs. 3B and C ) . The work of Rudall (195558) shows that the Lingula cuticle contains randomly oriented b- chitin fibrils and is capable of 50% expansion.

Both terebratulid and lingulid cuticles appear rather amorphous in the electron microscope. Irregular transverse fibrils of average diameter less than 5 nm can be seen in the TEM near the inner edge of the proximal Glottidia cuticle and TEM studies of the Terebratulina pedicle show that the cuticle is more dense at its external edge. The exterior of the Terebratulina cuticle appears smoother than that of Lingula in the SEM. The latter shows transverse fibres about 1 pm in diameter and an irregular concentric lay- ering. In Terebratulina . the layering is less obvious and the exterior shows discontinuous transverse folds. Towards the distal end of the rootlets the cuticle is replaced by a ring of long- itudinal fibrils.

Ultrastructure of the pedicle epithelium, rootlets and rootlet endings A transverse section through the main trunk of the pedicle of Terebratulina shows that the pedi-

LETHAM 11 (1978)

Fig. 3. Diagrams of the Lingula pedicle. 0 A. The pedicle seen through transmitted light. x 2. 0 B. The pedicle after removal of adherent sand grains. x 2. 0 C. Diagrammatic representation of the coelomic bulb.

LEXHAIA 1 1 (1978) Ultrastructure of the brachiopod pedicle 335

cle epithelium is on average 9-18 pm high (Fig. IA). The apical border of each epithelial cell next to the cuticle is irregular, with cytoplasmic projections about 0.25 pm high covered by a web of finely fibrillar material. This web may repre- sent material being secreted into the cuticle by the cell. The basal cell border is also irregular. Lateral cell borders are very tightly folded, and there may be dilated intercellular spaces. The folding of lateral cell membranes may provide a large surface area for secretion or it may allow cells to expand in height during changes in shape of the pedicle. Cellular organelles include tono- fibrils, electron-dense membrane-bound droplets on average 0.28 pm in diameter, clear vesicles about 0.4 pm in diameter, free ribosomes, a little rough endoplasmic reticulum and mitochondria. The Golgi apparatus is not well-developed.

Where the pedicle epithelium is joined to that of the mantle it is reduced in height and the apical border is a mass of clear vesicles about 0.5 Fm in diameter. The first four cells of the outer epithelium of the mantle seen in transverse section are joined to the periostracum which emerges as a layer about 0.5 pm thick. These cells show a better developedGolgi apparatus and smaller droplets and vesicles, and these features are prob- ably associated with the secretion of periostracal material.

A similar section through the GIottidia pedicle shows that the pedicle epithelium is about the same height as that of Terebratulina (Fig. IB). The ultrastructure of the epithelium is similar, although the apical cell border has larger cyto- plasmic projections, about 2 pm high, and the basal border is more regular. Lateral cell mem- branes are not as tightly folded and there may be dilated intercellular spaces at the cell base. There are more tonofibrils, larger dense droplets (on average 1.19 pm in diameter), smaller clear vesicles (on average 0.15 pm in diameter) and a more prominent Golgi apparatus.

The common features of epithelial cells of the main trunk of Glottidia and Terebratulina pedi- cles are therefore cytoplasmic projections on the apical border, folded lateral membranes, tono- fibrils, rough endoplasmic reticulum, electron- dense droplets and clear vesicles (Fig. 4A and B). These same features are also seen in outer epithelial cells of the mantle of Glottidia, but not in outer epithelial cells of Terebratulina which only show slightly folded lateral cell membranes, droplets and vesicles. Ultrastructural features common to the pedicle epithelial cells of Tere- bratulina and Glottidia and to the outer epithelial cells of Glottidia may be related to their common function of producing chitinous material. Cyto-

plasmic projections provide a large surface area which could be used for the secretion of cuticle components; similarly, folds in the lateral mem- branes increase surface area and rough endo- plasmic reticulum is involved in protein syn- thesis. Tonofibrils are thought to be cytoskeletal organelles of limited contractility; they may be necessary to provide support for cells adjacent to chitinous materials. The change in the cell surface of the pedicle epithelium of Terebratulina at its junction with the outer epithelium may represent a change in the production of the chitinous cuticle at this point.

Two types of Terebratulina pedicle rootlet sections can be found in the shells of Modiolus modiolus which form their substrate. In prox- imal rootlet sections of approximately 100 pm diameter the epithelium is about 3 pm high. Api: cal and basal cell borders are more regular than those of epithelial cells of the pedicle trunk, al- though cytoplasmic projections may still be seen (Fig. 5C). Lateral cell borders are less folded, tonofibrils and rough endoplasmic reticulum are absent and dense droplets are about twice as large as those of the trunk epithelium. Clear ves- icles are smaller (about 0.25 pm in diameter) and are found at the basal and apical cell borders. Otherwise ultrastructural features are similar to those of the pedicle trunk epithelium (Fig. 4C), except that the Golgi apparatus is better devel- oped and glycogen is present, suggesting that the secretory activity of these cells differs. The con- nective tissue core differs from that of the trunk as the collagen fibres are less densely packed and are interspersed with star-shaped electron- dense figures which may represent elastic fibres and small droplets of medium electron-density and averaging about 60 nm in diameter. The chi- tinous cuticle in these rootlets is absent; instead a layer of moderately electron-dense fibrous material forms the outer covering (Fig. 5A). This agrees with the finding that the thick cuticle does not extend to the surface in contact with the substrate (Bromley & Surlyk 1973). The fibres are on average 0.2 pm in diameter and run longitudinally.

More distal sections of the rootlets about 65 pm in diameter reveal that collagen fibres have disappeared from the central core. Instead, ag- gregations of droplets of electron-lucent material about 2.7 pm in diameter are interspersed with cellular processes (Fig. 5B). These processes show similar inclusions to those of the pedicle epithelium and are probably derived from them.

336 Sarah Mcickay and Roger A . Hewitt

C mitochondrion

vesicle I

\ I

glycogen I Golgi apparatus

D

LETHAIA 11 (1978)

B

microvilli

tonofibrils

Fig. 4. Diagrams to show variations of pedicle epithelial cells. 0 A. A typical epithelial cell of the pedicle trunk of Terebratuli- nu. 0 B. A typical pedicle epithelial cell ofc lof t id ia . 0 C. A typical Terebrafulina pedicle rootlet epithelial cell. 0 D. A typical microvillous cell of the Gloftidia pedicle epithelium at the pedicle ending.

Droplets similar to those of the core can be seen in some pedicle epithelial cells, being extruded into the centre. The pedicle epithelium produces fibrous material at both free cell surfaces; fi- brous masses bordering the central core have clear vesicles on average 0.19 pm in diameter dispersed between them. Epithelial cells are slightly higher than those of thicker rootlet sec-

tions. Ultrastructural differences are that larger cytoplasmic projections, on average 0.47 pm high, are restricted to the surface bordering the outer fibrous layer, suggesting that it is more active in secretion, and lateral cell surfaces are straight. There are both large moderately elec- tron-dense droplets of average diameter 1.63 pm and electron-lucent droplets of average diameter

LETHAIA I I (1978) Ultrastructure of the brachiopod pedicle 337

Fig. 5 . Electron micrographs of the Terebratulina rootlet. 0 A. Outer fibrous material of the rootlet, extending along the conchiolin walls of the substrate. ~ 3 6 0 0 . B. The central core of cellular material and electron-lucent material surrounded by inner fibrous material, epithelium and outer fibrous material. x 3600. 0 C. The rootlet epithelium between fibrous material (top left) and connective tissue (bottom right). x 15,000. 0 D. Clear vesicles at the rootlet periphery. x60,OOO.

0.65 pm. Clear vesicles are smaller, on average 0.17 pm in diameter.

Similar sized clear vesicles can occasionally be seen in the fibrous layer and smaller clear vesicles can also be seen in the fibrous material at the periphery of the rootlet. It is suggested that these vesicles are released from the epitheli- al cells of the distal rootlet and pass to the peri- phery where they may play a part in the break- down of the Modiolus shell fabric. Similar ves-

icles have been associated with the resorption of bone by osteoclasts (Lucht 1971, 1972, 1973). These vesicles are comparable in size and appearance to those of the Terebratulina rootlet (Fig. 5D). Osteoclast vesicles have been shown to contain acid phosphatase and are thought to be primary lysosomes. These vesicles can pass to the extracellular space between the osteoclast and bone. The Golgi apparatus is known to par- ticipate in the formation of primary lysosomes

338 Sarah Mackay and Roger A . Hewitt LETHAL4 1 I (1978)

Fig. 6. Electron micrograph showing the Modiolus periostra- cum with holes tilled by microbenthos and material from the rootlet of the Terrbratdina pedicle seen at the top left cor- ner. ~ 3 6 0 0 .

and it is interesting that it is better developed in the epithelial cells of rootlets than in those of the pedicle trunk.

At the junction with the Modiolus shell the rootlet fibres become more closely packed to- gether and in the TEM are seen as a homogene- ous electron-dense mass extending along the conchiolin walls of the nacreous layer of the substrate. There may be a lighter zone within the periphery of the rootlet of less densely-packed fibres. The rootlet margin seen in the SEM shows a zone of granules about 0.2 pm in diame- ter extending up to 6.0 pm into the nacre of the Modiolus. The adjacent unaltered laminar nacre consists of the typical eight-sided aragonite ta- blets bounded by a sheath of acid resistant struc- tural protein (conchiolin) (Wilber 1972: 115).

The outer margin of the granular layer shows a relief up to 2 pm between adjacent aragonite

tablets. Since this distance is less than the length of the tablets, it seems that total dissolution of the aragonite does not immediately follow the destruction of the conchiolin walls. This view is supported by the observation of horizontal clus- ters of granules within the granular layer.

The fibrous material of the rootlet appears to extend its infiltration into the Modiolus shell fab- ric along the conchiolin walls in TEM sections (Fig. SA).This finding supports the suggestion by Travis andGonsalves (1969) that the sequence of events during the breakdown of molluscan sub- strates involves a primary attack on the organic matrix by lysosomal hydrolytic enzymes and a secondary attack on the inorganic components.

Rootlets can penetrate the periostracum of the Modiolus valve in the same way. The periostra- cum appears as a homogeneous electron-dense layer with holes filled both by extensions of the microbenthic settlement on the exterior of the periostracum and by pedicle rootlet material (Fig. 6). The rootlet material is composed of densely packed fibres which may be more loose- ly packed at the periphery of the rootlet, forming a fine network rather than fibrous bundles. Also seen at the periphery are electron-dense droplets of average diameter 0.33 pm which may repre- sent decomposed periostracal material and clear vesicles of average diameter 0.18 pm similar to those of the distal rootlet.

Modifications also occur at the Glottidia pedi- cle ending and these are probably associated with its ability to collect sand grains. The outer layer of chitinous cuticle disappears and the mu- scular layer is reduced to half its thickness in the trunk. Some groups of pedicle epithelial cells bear microvilli at the apical cell surface on av- erage 3.6 pm high. Ultrastructural features are similar to those of the pedicle epithelium of the trunk. Cells may have dilated intercellular spa- ces basally, with clear vesicles of average diame- ter 0.47 pm in the spaces. An impersistent mu- copolysaccharide film covers the apical surface (Fig. 4D). Rough endoplasmic reticulum is more plentiful and is often found in the folds of the lateral cell membranes. The cells contain glyco- gen, a few small electron-dense droplets on av- erage 0.27 pm in diameter and clear vesicles twice as large as those in pedicle epithelial cells of the trunk. Some cells also show larger droplets on average 1.13 pm in diameter. Other epithelial cells show similar inclusions but lack a microvil- Isus border, the apical edge is instead extended into cytoplasmic protrusions on average 0.6 pm

LETHALA 1 I (1978) Ultrastructure of the brachiopod pedicle 339

long. The microvillous border of these cells has presumably been worn off by wear against the sand grains during the removal of the brachiopod from its burrow.

The large electron-dense droplets seen in some epithelial cells of the pedicle ending of Glottidia appear to be mucous droplets; they may be extruded to the outside and spread around the pedicle tip by microvillous action. The mucous secretion could act as a glue to bind the sand grains, forming a firm base from which the brachiopod could lever itself up as described by Thayer & Steele-PetroviC (1975). The cross sectional shape of the pedicle changes to an undulated circle at the tip and this may be a modification of shape to trap sand grains be- tween the ridges so formed.

The author of a paper on the structure of the gonads and pedicle of articulate brachiopods (Haro 1963) comments that the pedicle has the same basic structure as the mantle but that colla- genous fibres delimit lacunar spaces containing capsules with syncytial elements resembling car- tilage. The results of electron microscopy do not support these light-microscopical findings; such lacunar spaces have not been seen and the con- nective tissue of the pedicle trunk and rootlets of Terebratulina shows no resemblance to car- tilage.

Concluding remarks The lingulid and terebratulid pedicles differ greatly in functional significance and it seems likely that both types evolved early in brachio- pod phylogeny. In early Cambrian times, sessile brachiopods were presumably either attached by mucous secretions from the distal end of the pedicle or had a short unattached pedicle that served as a lever as in the Recent Magadina arningi (Richardson & Watson 1975). In lingulids this tendency towards mobility was increased to the point where the shell could survive in areas of fast sedimentation although this development may have occurred some time after the emer- gence of the group.

It seems likely that borings may have been produced by many extinct articulate brachio- pods (see Thayer 1977:397). Their recognition may be far from easy. The studies on Terebratu- lina indicate that pedicle rootlets can break

down organic matrices and thus bore into arago- nite, calcite, carbonate-apatite and entirely organic substances. The borings must be deep enough to afford anchorage and wide enough to accommodate the pedicle epithelium which appears to be necessary for boring.

Such borings have been identified on the shell of the Miocene species Terebratula terebratula and have been reported on Lower Cretaceous substrates by Bromley & Surlyk. Now that they are becoming better known it is hoped that they can be confirmed for older stocks.

Acknowledgements. -We wish to thankDr. A. Williams of the University of Glasgow for his helpful comments on the man- uscript. We are also indebted to Dr. H. M. Pedley for provid- ing fossils from sample 396 of his Table 1 (Pedley 1976).

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Thayer, C. W. 1977: Strength of pedicle attachment in articu- late brachiopcds: ecological and paleoecological signifi- cance. Paleobiology I, 388-399.

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