Fine structure of Merkel cells in lampreys

Summary The structure of Merkel cells occurring in the epidermis of adult and larval stages of Lampetra spp. is described; it is comparable to that reported from the gnathostome classes. The cells bear microvilli, grouped on the distal and proximal aspects, and are associated with sparsely branching and varicose nerve fibres. One branch of the neurite bears a spur-like process which indents the proximal side of the Merkel cell. Most of the specific Merkel granules are situated in the vicinity of this neurite projection; the cell membrane adjacent to the tip of the spur process bears structures resembling presynaptic densities. Occasionally, desmosome-like junctions are found between the neurite and the Merkel cell.The authors thank the Fresh Water Biological Association and the Department of Biological Sciences, University of Bath, for supplying the material, Dr. H. Fox for giving some prepared blocks of Lampetra planeri adults, Mr. B.L. Pirie for technical assistance, and the Science Research Council for support through grant GR/A/3740.6     

Ultrastructure of Merkel corpuscles in the tongue of the finch,Lonchura striata

Summary Merkel corpuscles in the lingual mucosa of the finch, Lonchura striata, were examined by means of the argyrophilic reaction and electron microscopy. These corpuscles are composed of 12 to 20 flattened Merkel cells and enclosed nerve terminals. The present study demonstrated for the first time argyrophilia in avian subepithelial Merkel cells with the use of Grimelius silver stain. Electron-microscopically, the Merkel cell was characterized by the presence of numerous densecore granules, approximately 80 to 140 nm in diameter, as well as specialized contacts with nerve terminals. The granules showed a tendency to accumulate in the cytoplasm in close association with both nerve terminals and basal lamina. This study also provided unequivocal evidence for exocytotic discharge of Merkel-cell granules at the plasma membrane facing not only the nerve terminals but also the basal lamina. The exocytotic figures toward the nerve terminals can be regarded as synaptic discharge of Merkel-cell granules, but the possibility also exists that the Merkel-cell granules may exert a trophic effect on the nerve terminals. The exocytotic release of Merkel-cell granules toward the basal lamina with no relation to nerve terminals may suggest an endocrine (paracrine) function for the Merkel cell. The avian subepithelial Merkel cells qualify as paraneurons, but their exact nature and function remain enigmatic as is the case of intraepithelial Merkel cells in other vertebrates.     

Some cytologic characteristics of the hemocytes of Limulus during clotting

The hemolymph of the horseshoe crab, Limulus (Xiphosura) polyphemus, contains a single cell type. The hemocytes are ovoid and contain many refractile granules. One-half to one minute after the onset of clotting the hemocytes swell and a hyaline cytoplasmic ring, essentially devoid of granules, appears about their circumference. During this time the granules disperse and the nucleus becomes visible. Three to five minutes following the initiation of clotting, the cell extends long pseudopodial processes. Phase contrast time-lapse cinematography reveals that the cells are extremely motile during this phase. Concomitant with these changes, many of the granules lose their refractility and one by one disappear from the cytoplasm leaving what appears to be a vacuole. Electron micrographs of native (un-clotted) hemocytes and of clots fixed in glutaraldehyde and post-fixed in osmium reveal that the membrane-bounded granules of native cells are very dense and homogeneous with no evidence of an internal structure. One-half to one minute after clotting, however, they become less dense and 250 Å microtubules spaced at ca. 500 Å intervals appear parallel to the long axis of the granule. Further degradation of the granule ensues and involves (a) change to a spherical shape, and (b) further decrease in density and better resolution of the microtubules. The microtubular component subsequently disintegrates leaving a membrane-bounded granule containing particulate material. Finally, the membrane of the granule fuses with the plasma membrane and the components of the granule are dispersed in the plasma where they presumably contribute to the formation of the gelatinous clot.     

Small granulated cell types in rat superior cervical and coeliac-mesenteric ganglia

Summary In the rat superior cervical and coeliac-mesenteric ganglia we have observed three types of small granulated (SG) cell: Type I cells are characterised by membrane-bounded cytoplasmic granules with a core of variable, moderate to low electron-density, whose limiting membranes are rounded in profile ranging from 50–150 nm in diameter. Type II SG cells contain numerous highly electron-dense, polymorphic cytoplasmic granules ranging from 100–300 nm in diameter. The haloes of Type II cell granules are variable in shape, and the core is often eccentrically located or fragmented. Type III SG cells contain membrane-bounded granules with a core of variable moderate to low electron-density. In profile these granules appear oblong or circular with average dimensions of 170 × 50 nm. All three SG cell types receive cholinergic-type pre-ganglionic terminals whose afferent nature is confirmed by their degeneration following pre-ganglionic neurectomy. Only Type I cells have been observed to donate efferent synapses to dendrites of principal ganglionic neurones and are thus interneuronal.This work was in part supported by a grant from the Medical Research Council. We wish to thank Mr. T.T. Lee for valuable technical assistance and Mr. P.F. Hire and Mr. K. Twohigg for illustrative help     

Electron microscopic study on the thyroid gland of the salamander Hynobius nebulosus in the breeding season

Summary The thyroid gland of adult salamanders, Hynobius nebulosus, in the breeding season was studied by electron microscopy. The follicular cells are different in cell height and fine structures; the taller cells with many cell organelles and granules and the lower cells with a few cell organelles and granules are both present in the same follicle. In the cytoplasm, three types of membrane-bounded granules, namely, cytosomes, colloid droplets, and vacuolar bodies and circular membrane complexes occur. The vacuolar bodies are subdivided into two types; the ordinary type having loosely distributed particles and the specific type containing tubules and/or closely packed filaments, crystalloid structures, except for the particles. The chromophobe colloids within the Bensley-cells correspond to extremely large, ordinary type vacuolar bodies, while the Langendorff-colloid cells possess increased numbers of granular cisternae of endoplasmic reticulum and a ribosome-rich, dense cytoplasmic matrix but not extremely large colloid. The intracytoplasmic circular membrane complexes appear in the Golgi area of cytosome-rich cells. It is suggested that they originate from the Golgi apparatus which was activated to produce many cytosomes. Intranuclear inclusions consisting of microtubules and filaments and tight junctions between two adjacent lateral plasma membranes are occasionally encountered.     

On the ultrastructure of polypeptide hormone-producing cells in the gut of the ascidian,Ciona intestinalis L. and the Bivalve,Mytilus edulis L.

Summary Ultrastructural evidence has been found for the presence of polypeptide hormone-producing cells in the gut of Ciona intestinalis L. and Mytilus edulis L. which do not appear to have been described before. Due to their localization and ultrastructural characteristics, it is suggested that the cells in Mytilus edulis probably produce an insulin-like substance and that some of these cells in Ciona intestinalis may produce 5-HT (5-Hydroxytryptamine). In each species only one granulated cell type can be observed. The granules, which are electron dense and membrane bound, also show a halo. The average diameter of the granules is 100–200 nm for Ciona and 200–400 nm for Mytilus.I thank Mr. G. Bargsten, M.A., Dept. of Marine Zoology, University of Kiel, for the supply of the animals     

Whale tear glands in the bowhead and the beluga whales: Source and function

Orbital glands are found in many tetrapod vertebrates, and are usually separate structures, consisting of individual glands lying in the eyelids and both canthi of the orbit. In cetaceans, however, the orbital glandular units are less distinct and have been described by numerous authors as a single, periorbital mass. There are few histochemical and immunhistochemical studies to date of these structures. In this study, we examined the orbital glandular region of both the bowhead whale (Balaena mysticetus: Mysticeti) and the beluga whale (Delphinapterus leucas: Odontoceti) using histological, histochemical, and immunohistochemical techniques. Histologically, in the bowhead, three glandular areas were noted (circumorbital, including Harderian and lacrimal poles), palpebral (midway in the lower eyelid), and rim (near the edge of the eyelid). In the beluga, there was only a large, continuous mass within the eyelid itself. Histochemical investigation suggests neither sexual dimorphism nor age-related differences, but both whales had two cell types freely intermingling with each other in all glandular masses. Large cells (cell type 1) were distended by four histochemically distinct intracellular secretory granules. Smaller cells (cell type 2) were not distended (fewer granules) and had two to three histochemically distinct intracellular secretory granules. The beluga orbital glands had additional lipid granules in cell type 1. Counterintuitively, both lipocalin and transferrin were localized to cell type 2 only. This intermingling of cell types is unusual for vertebrates in whom individual orbital glands usually have one cell type with one to two different secretory granules present. The heterogeneity of the orbital fluid produced by cetacean orbital glands implies a complex function, or series of functions, for these orbital glands and their role in producing the tear fluid.     

Species variability in the expression of met- and leu-enkephalin-like immunoreactivity in mammalian Merkel cell dense-core granules

Summary Immunogold staining failed to show met-enkephalin immunoreactivity in the Merkel cell dense-core granules of rats when examined by electron microscopy, but showed gold particle staining in the Merkel cell dense-core granules of mice and nude mice. Merkel cells of hamster, guinea pig, rabbit, cat and dog were also examined using a similar method, and different antisera dilutions. Immunogold particles were consistently found in the dense-core granules of mice and nude mice at all antisera dilutions, but not in the other species, except in the dog, where a very low labelling response was encountered. Merkel cells from skin touch domes or sinus hair follicles, did not exhibit any difference in peptide expression as far as met-enkephalin immunoreactivity was concerned. In addition, all species studied, including mice and nude mice, did not show leu-enkephalin immunoreactivity in their Merkel cell dense-core granules. It is concluded that species variability in peptide expression occurs in the Merkel cell dense-core granules, and may be closely related to the different methodologies used.     

Fine structure of the adenohypophysis in immature sockeye salmon,Oncorhynchus nerka

Summary The ultrastructure of the secretory cells of the adenohypophysis of juvenile sockeye salmon was investigated. Pituitary glands were collected from immature fish transferred experimentally to sea water and subsequently returned to fresh water. The rostral pars distalis contained three cell types: ACTH cells, prolactin cells, and non-secretory cells. The prolactin and non-secretory cells were joined together in the form of follicles by desmosomes and they both had cilia and microvilli projecting into the follicle lumen. Various follicular structures such as lumen, multivesicular structures, and peripheral basement membrane are discussed as possible sites of prolactin cell granule release. The columnar ACTH cells were found at the junction of the rostral pars distalis and the neurohypophysis. The cytoplasmic granules in these cells were characteristically separated from their limiting membrane by a clear space. Multivesicular structures were also found in association with this cell type. The caudal pars distalis also contained three cell types: one acidophil (putative somatotrop) and two basophils (putative thyrotrops and gonadotrops), all of which were similar to those described in adult fish. The pars intermedia contained only one cell type. They appeared to be active cells and were characterized by containing membrane-bounded granules similar to those found in the ACTH cells. Changes in ambient salinity had no apparent effect on any cell type described.The work was supported by a grant in aid of research from the National Research Council of Canada. We wish to thank Mr. R. Lindsay, Mr. C. Cooper, and Mr. G. Longworth for their technical assistance. We would also like to thank Mr. S. Killick of the International Pacific Salmon Fisheries Commission for his assistance in the collection of fish and Dr. H. Cook for his helpful discussion of the project. This paper is No. 058 in the University of Guelph Migration Series.     

Ultrastructure of Merkel corpuscles and so-called "transitional" cells in the white Leghorn chicken

In the chicken Merkel corpuscles are located in the dermis and consist of specialized Merkel cells, discoid nerve endings and lamellar cells. Merkel cells contain characteristic membrane-bound dense-core granules and bundles of microfilaments. Asymmetric junctions, synapse like, with thickened membranes and clusters of dense-core vesicles were observed between the Merkel cells and the nerve endings. The nerve ending is derived from myelinated nerves and sometimes contains clusters of clear vesicles. A laminar system formed by lamellar cells of the Schwann cell type encloses the Merkel cells and the nerve endings. So called "transitional" cells, showing some of the morphological features of both keratinocytes and Merkel cells, were observed in the basal layer of the epidermis. One was located partly in the epidermis and partly in the dermis. The structure of Merkel corpuscles is compared with that of Merkel cells in other tetrapods. The developmental significance of "transitional" cells and the origin of Merkel cells are discussed.     

Neuron-specific enolase and serotonin in the Merkel cells of conger-eel (Conger conger) epidermis

Summary Immunocytochemical techniques were used to investigate the distribution and co-localization of neuronspecific enolase (NSE) and serotonin (5-HT) in the skin of the conger eel, Conger conger. NSE and 5-HT immunoreactivity were found in Merkel cells; these cells were also identified at the electron-microscope level by the presence of characteristic granules and their association with an intraepithelial nerve ending. For the first time, it was demonstrated that Merkel-cell granules of vertebrate skin exhibit in immunoreaction with 5-HT. The production of amines may indicate that the Merkel cells of C. conger have both secretory capabilities and transduction functions.However, immunocytochemical investigation of the synaptic zones at the electron microscope level will be necessary to confirm this hypothesis.The present histochemical results suggest that NSE and 4-HT may be marker substances for Merkel cells, and that immunocytochemistry is a useful tool for the light-microscopic localization of these cells.     

On the occurrence of argyrophil cells in salivary glands

Summary Salivary glands (parotid, submandibular and sublingual glands) of nine mammalian species were investigated with respect to presence and localization of argyrophil and argentaffin cells. With the exception of the parotid gland of the rat, no positive staining was observed within the examined glands. In the rat parotid distinctly argyrophil cells could be demonstrated in the intercalated ducts. Histochemical studies of the cells, ultrastructural analysis of their cytoplasmic granules as well as their reactions to certain drugs indicate that these cells are of exocrine rather than of endocrine nature. After a subcutaneous injection of pilocarpine, the intensity of the argyrophil staining was markedly reduced. No specific catecholamine fluorescence could be detected within the cells, not even after pretreatment of the animals with high doses of L-DOPA. The membrane-bounded cytoplasmic granules of the intercalated duct cells furthermore displayed a strong positive staining reaction after treatment of ultrathin Vestopal sections with the periodic acid-chromic acid-silver technique of Rambourg et al. (1969).Supported by grants from the Swedish Medical Research Council (Project No. 12X-718), and the Medical Faculty of the University of Umeå. The skilful technical assistance of Miss Siw Domeij is gratefully acknowledged     

Electron microscopy of the leucocytes of the typhlosole in ammocoetes,with special attention to the antibody- producing cells

In an attempt to determine the role in the immune responses of the typhlosole, a hematopoietic tissue along the ventral wall of the larval lamprey Lampetra reissneri, scanning and transmission electron microscopic observations were made on ammocoetes hyperimmunized with sheep red blood cells. Besides including the cells of the erythrocyte series, this tissue also contained the following leucocytes forming an amorphous parenchyma: the cells of the granulocyte series, the most predominant cell type, possessing a markedly lobed nucleus and membrane-bounded granules of various sizes; the macrophages possessing primary and secondary lysosomes and long lamellipodia on the cell surface; the lymphocytes of a large nucleocytoplasmic ratio with a number of long, spiky microvilli, constituting a major type of rosette-forming (antigen-binding) cells; and the plasma cells possessing highly extended cisternae of rough endoplasmic reticula that are characteristic of the higher vertebrates. The immunoperoxidase technique, which employs rabbit antibodies against lamprey immunoglobulin, proved that these plasma cells do contain immunoglobulin. These results strongly indicate that the typhlosole of the larval lamprey, besides functioning as a hematopoietic tissue, is actively involved in the antibody responses. It is also stressed that the plasma cell occurs in the most primitive vertebrates as an immunologically competent cell.     

Cell types of the endocrine pancreas in the shark Scyliorhinus stellaris as revealed by correlative light and electron microscopy

Summary In the pancreas of Scyliorhinus stellaris large islets are usually found around small ducts, the inner surface of which is covered by elongated epithelial cells; thus the endocrine cells are never exposed directly to the lumen of the duct. Sometimes, single islet cells or small groups of endocrine elements are also incorporated into acini. Using correlative light and electron microscopy, eight islet cell types were identified:Only B-cells (type I) display a positive reaction with pseudoisocyanin and aldehyde-fuchsin staining. This cell type contains numerous small secretory granules (Ø280 nm). Type II- and III-cells possess large granules stainable with orange G and azocarmine and show strong luminescence with dark-field microscopy. Type II-cells have spherical (Ø700 nm), type III-cells spherical to elongated granules (Ø450 × 750 nm). Type II-cells are possibly analogous to A-cells, while type III-cells resemble mammalian enterochromaffin cells. Type IV- cells contain granules (Ø540 nm) of high electron density showing a positive reaction to the Hellman-Hellerström silver impregnation and a negative reaction to Grimelius' silver impregnation; they are most probably analogous to D-cells of other species. Type VI-cells exhibit smaller granules (Ø250 × 500 nm), oval to elongated in shape. Type VI-cells contain small spherical granules (Ø310 nm). Type VII-cells possess two kinds of large granules interspersed in the cytoplasm; one type is spherical and electron dense (Ø650 nm), the other spherical and less electron dense (Ø900 nm). Type VIII-cells have small granules curved in shape and show moderate electron density (Ø100 nm). Grimelius-positive secretory granules were not only found in cell types II and III, but also in types V, VI, and VII. B-cells (type I) and the cell types II to IV were the most frequent cells; types V to VII occurred occasionally, whereas type VIII-cells were very rare.This work was supported by a fellowship from the Ministry of Education of Japan and the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg (La 229/8)     

Cytodifferentiation in the accessory glands of Tenebrio molitor. XI. Transitional cell types during establishment of pattern

The bean-shaped accessory glands of male Tenebrio consist of a single-layered epithelium which is surrounded by a muscular coat. The epithelial layer, which produces precursors of the wall of the spermatophore, contains eight secretory cell types. Each secretory cell type is in one or more homogenous patches, and discharges granules which form one layer of the eight-layered secretory plug. Maturation begins in cell types 4, 7, and 6 on the last pupal day. A newly identified cell (type 8) in the posterolateral epithelium matures last. Cells of individual types mature in synchrony, and their secretory granules “ripen” in a sequence that is characteristic for each type. As the secretory cells of each patch mature, unusual short-lived cells appear at interfaces between patches. In some cases the secretory granules in these boundary cells have ultrastructural features which are mixtures of the definitive characteristics of granules in adjacent cell types. The transitional cell types disappear at 3–4 days after eclosion. Intermediate cell types are absent in the mature gland and boundaries between the patches are distinct. The transitional cells may form granules of intermediate structural characteristics as a dual response to cellular interaction with adjacent and previously differentiated secretory cells.     

Localisation of VIP- and CGRP-like substances in the skin and sinus hair follicles of various mammalian species

Using an ultrastructural postembedding immunogold technique, we demonstrated vasoactive intestinal polypeptide (VIP)- and calcitonin gene-related peptide (CGRP)-like immunoreactivity in the Merkel cell dense-cored granules of skin and sinus hair follicles of adult cat and dog. The VIP-like substance was located in cat Merkel cells while both VIP- and CGRP-like substances were colocalised in dog Merkel cells. In cat Merkel cells, the magnitude of labelling of VIP was qualitatively higher than in dog Merkel cells. In the dog Merkel cell, CGRP appeared as the most abundant peptide. Dense-cored granules were labelled for these peptides. In addition, mast cells encountered in the dermal region of dog skin were also found to be immunolabelled by VIP antiserum. The immunoreaction was found to be confined to the secretory granules of the cells. Furthermore, all non-myelinated nerve plexuses encountered in the dermal region of the skin and the sinus hair follicles of the various mammalian species studied were immunolabelled by CGRP antiserum. The specific location was again restricted to the dense-cored granules present in these nerves. As VIP and CGRP have potent vasodilatory effects, our observations suggest that Merkel cells may play a separate or synergistic role in regulatory functions of the skin neuroendocrine cell, exerting their influence by paracrine, endocrine and neurocrine pathways, or a combination of these. Different methodologies of double labelling with different sizes of gold particles are also discussed.     

Electron microscopic studies of the corpuscles of Stannius of an airbreathing teleost (Heteropneustes fossilis)

The ultrastructure of the corpuscles of Stannius (CS) ofHeteropneustes fossilis reveals a homogenous cellular composition characterized by only one cell type, with large secretory granules and abundant ribosomal endoplasmic reticulum. These cells are comparable to the type 1 cell described in the CS of other teleosts; type 2 cells, whose presence is ubiquitous in the CS of freshwater species are absent inH. fossilis. Our data on the CS ofH. fossilis demonstrate that not all freshwater species possess type 2 cells in their CS and these are not essential for life in freshwater.     

The fine structure of the light organ of the glowworm Lampyris noctiluca

Summary The four main parts of the glowworm light organ are the cuticle, the hypodermis, the photocyte layer and the reflector cell layer. The hypodermis is one cell thick and it contains hypodermic glands. These glandular cells have a lumen that opens to the outside of the cuticle. Projecting into the lumen are numerous microvilli. Between the hypodermis and photocytes are typical insect tunicated nerve fibres. They pass down between the photocyte and reflector layer cells. They do not appear to innervate the photocytes and they are thought to innervate adjacent muscle fibres or to be sensory. Tracheoles are commonly present between the photocytes but no tracheolar end organs are found. The photocytes contain amorphous granules, mitochondria, photocyte granules and a vesiculated reticulum. All, except the mitochondria, are absent from the reflector layer and so probably have some connection with light production. The reflector layer contains glycogen granules, clear spaces thought to be the sites of urate crystals, and membranous granules. The latter granules are sometimes found in photocytes adjacent to the reflector layer whilst amorphous granules are sometimes absent from these adjacent cells. So a cell layer with some features of the photocyte and reflector layer cells is present. These morphological findings are discussed with regard to the unknown function of the reflector layer and the control of light emission. Acknowledgments. We would like to thank Professor J. Z. Young and Dr. E. G. Gray for their advice and encouragement, Mrs. Jane, Astafiev for drawing fig. 1, Mr. S. Waterman for photographic assistance, Miss Cheryl Martin for secretarial assistance, and many colleagues for help in collecting specimens of glowworms.     

Ultrastructure of the neurohypophysis of the teleost Poecilia latipinna in relation to neural control of the adenohypophysial cells

Summary The structure of the neurohypophysis of Poecilia latipinna (green molly, sailfin molly) was studied with the electron microscope. Profile diameters of neurosecretory granules in the non-myelinated neurohypophysial nerve fibres were measured and mathematically corrected for error due to section thickness. Six different types of nerve fibres could be distinguished by statistical classification of their granules and by other ultrastructural features. One fibre-type (type B) contained granules with a mean diameter of 85 nm, and the other five types (types Ala, Alb, A2, A3 and A4) all contained granules with mean diameters greater than 100 nm. Synaptic contacts were observed between type B fibres and all the adenohypophysial cell-types, although in the case of the ACTH cells the synapses were separated from the cell membrane by a continuous double basement membrane. Type A fibres were observed to contact the cells of the proximal pars distalis and pars intermedia, but did not form synapses. However, synapses occurred between type A fibres and pituicytes, and between type A fibres and the pericapillary basement membrane in the interior of the neurohypophysis. The possible roles of the different types of nerve fibres in controlling the adenohypophysial cells are discussed in the context of evidence from other teleosts.We thank Mr. W.A. Thomson and Mr. D.I. Hollingworth for technical assistance, and Dr. D.I.C. Pearson (Department of Physics, University of Nancy, Nancy, France) for advice on mathematical analysis and computer programs. The work was carried out during the tenure of an S.R.C. Research Studentship by T.F.C.B.     

Hemocytes of the palaemonids Macrobrachium rosenbergiiand M. acanthurus,and of the Penaeid Penaeus paulensis

The hemocytes of two palaemonids and one penaeid were characterized using light and transmission electron microscopy (TEM). The blood cells in all three species were classified as hyaline hemocytes (HH), small granule hemocytes (SGH), and large granule hemocytes (LGH). The HH are unstable hemocytes with a characteristic high nucleo-cytoplasmic ratio. Their cytoplasm appears particularly dense and has from few to numerous granules that often exhibit a typical striated substructure. In both palaemonids, the great majority of the HH contain numerous granules, whereas in Penaeus paulensis, a small number of these cells have few or no granules. The cytoplasm of some HH of the penaeid exhibits typical electron-dense deposits. The granulocytes, LGH and SGH, contain abundant electron-dense granules that are usually smaller in the SGH. In both hemocyte types, the cytosol, but not the granules, is rich in carbohydrates (PAS positive) and numerous vesicles contain acid phosphatase (Gomori reactive). In all studied shrimps, the SGH and LGH were actively phagocytic when examined on blood cell monolayers incubated with the yeast Saccharomyces cerevisiae. A few mitotic figures (less than 1%) were observed in the granulocytes of P. paulensis, but not in the palaemonids. SGH is the main circulating blood cell type in both palaemonids, whereas HH is predominant in the penaeid. Based on morphological and functional features, it appears that the hyaline and the granular hemocytes of the three shrimp species represent different cell lineages. J. Morphol. 236:209–221, 1998. © 1998 Wiley-Liss, Inc.