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Ultrastructure of the foregut and associated glands of Calicotyle kröyeri (Monogenea: Monopisthocotylea)
ULTRASTRUCTURE
OF THE FOREGUT AND ASSOCIATED
GLANDS
OF CALZCOTYLE
KRGYERZ
(MONOGENEA : MONOPISTHOCOTYLEA) D. W. HALTON and S. D. STRANOCK Department
of Zoology,
The Queen’s (Received
University,
Belfast
28 Junuary
BT7 1NN,
Northern
Ireland
1976)
Abstract--HALToN D. W. and STRANOCK S. D. 1976. Ultrastructure of the foregut and associated glands of Calicoryle krGyeri (Monogenea: Monopisthocotylea). International Journal fur Parasitology 6: 517526. The mouth, pharynx and oesophagus of Calicotyle are lined by syncytial epithelia, and there are numerous unicellular glands associated with the oesophagus. An infolding of unmodified external tegument lines the mouth cavity and is connected by discrete cytoplasmic processes to subjacent perikarya. It contains two types of secretory body and its luminal surface is invested with a finely filamentous coating. The pharynx and oesophagus are lined by irregularly-folded epithelia that are interconnected by a septate desmosome. Membranous inclusions distinguish the pharynx epithelium and there is a w-ell developed basal lamina for insertion of the pharyngeal muscles. The oesophagus epithelium is perforated by the openings of the oesophageal glands. These lie in the surrounding parenchyma and produce a dense, membrane-bound secretion which is conveyed by duct-like extensions of the glands to the oesophagus lumen. The ducts are supported in places by microtubules and are anchored to the oesophageal epithelium by septate desmosomes. A septate desmosome also marks the junction between the epithelium and the gut caeca.
INDEX KEY WORDS: Calicotyle alimentary tract; gland cells.
krdveri;
monogenean;
microscopy,
INTRODUCTION
The
anterior part of the alimentary tract of consists of a mouth (surrounded by an oral sucker), pharynx and oesophagus, each of which is lined by a separate epithelium. An extensive collection of unicellular glands are situated on either side of the oesophagus and open into its lumen. Immediately behind the gland cell openings the gut divides into the two blind-ending intestinal caeca. C. kriiyeri
Mouth
cavity
The mouth is lined by tegument which is continuous with that covering the external body surface. Its general organization corresponds to that found in other parasitic platyhelminthes in that there are nucleated secretory regions--tegumentary cell bodies or perikarya-positioned deep in the parenchyma which connect by discrete cytoplasmic processes to an anucleate surface syncytium. This outer syncytial layer in Crrlicotyle is approx. 1.5-2 pm in thickness and is penetrated at fairly regular intervals by a series of surface infoldings or pits (Fig. 1). The pits are generally quite narrow in diameter and measure approx. 0.4 pm in depth. Morphological evidence of endocytosis, such as coated invaginations and related vesicles, was not observed. The plasmalemma of the tegument is asymmetric and displays apparent
digestion.
The work forms part of a comparative electron microscope study of gut structure in skin-feeding monopisthocotylean and blood-feeding polyopisthocotylean monogeneans which, it is hoped, will form a base-line for physiological investigation of nutrition in these parasites. AND
ultrastructure;
RESULTS
Cdicotyle kriiyeri inhabits the cloaca and rectum of skate and ray and feeds on desquamated epidermal cells and mucus. The food is digested by a combination of extraand intracellular processes, involving oesophageal glands and the caecal epithelium (Halton & Jennings, 1965). Ultrastructural studies by Halton & Stranock (1976) have already shown the caecal cells to be involved in endocytosis and intracellular digestion of mucus and epidermal cell debris. The present observations on the foregut and associated glands expand these studies to include morphological detail of the elements responsible for the extracellular phase of
MATERIALS
electron;
METHODS
Details of the collection of specimens of C. krtiyeri and their preparation for examination by electron microscopy have been given elsewhere (Halton & Stranock, 1976). 517
518
D.W.
HALTON~~~S.D.STRANOCK
continuity with a glycocalyx-like, filamentous coat (Fig. I, inset). Beneath the membrane there is a dense subsurface layer of finely fibrous material below which the tegument matrix is less dense and contains mitochondria and numerous dense secretory bodies. The mitochondria are oval or elongate in outline with moderately-dense matrices and relatively few transverse cristae. Although present in most parts of the tegumentary unit, the majority of mitochondria are concentrated near the basal plasma membrane. Two types of secretory inclusion body can be recognized in the tegument. The most numerous are membrane-bound spheres measuring between 0.15 and 0.3 pm in diameter and displaying a packed granular content (Fig. 1). They are present in all areas of the tegument and are formed in the subjacent ceil bodies. A number of them display increased granulation of content in the surface tegument and, in some instances, appear to release their content to the cytoplasm (Fig. 1, inset). The other inclusion bodies are rod-shaped and contain a central core of dense material (Fig. I). They measure approx. 0.1 pm in diameter and vary in length, appearing either straight or slightly curved. Their origin is unclear since they have not been observed in any of the tegumentary cell bodies examined. The cell bodies can be identified by the presence in their cytoplasm of the spherical tegumentary inclusion bodies in various stages of development (Fig. 2). The nucleus is large and ovoid and there are long narrow profiles of GER and numerous Golgi stacks involved in the formation of the secretion. The plasma membrane is irregular in outline and close junctions with adjacent parenchyma1 cells are fairly common. The basal plasma membrane of the surface tegument is frequently folded, forming short, slotlike invaginations of fairly constant width (30 nm) (Fig. 1). They occur singly or in groups of three or four in parallel array. Larger invaginations of variable width are also present and contain material that is continuous with the underlying basal lamina. These membranous configurations are usually branched and often extend as far as the apical cytoplasm. The basal lamina comprises a layer (approx. 0.1 pm in thickness) of finely fibrous material underneath the basal plasma membrane but separated from it by a narrow gap (Fig. 1). Below this, thick fibrous interstitial material fills all the available space between the tegumentary unit and the muscle fibres of the oral sucker. In places, this sheath of fibrous material is penetrated by spike-like insertions of the basal lamina. Pharynx
The pharynx is a large, well-developed muscular structure measuring approx. 450 pm in length and
1.J.P. VOL.6.1976
350 wrn maximum width. Its lumen is triradiate and fined by an apparently syncytiaf epithelium, measuring some 1.53 pm in height (Fig. 3). In all the specimens examined, the pharynx appears constricted so that its fining is highly folded, with undulant and frequently branched surface invaginations penetrating deep into the cytoplasm. The free surfaces of the infolded membrane are closely apposed producing a dense, multilaminate appearance in section (Figs. 3 & 4). The basal plasma membrane is also infolded, with broad finger-like invaginations of membrane extending in curved profiles for some distance into the cytoplasm. In places the invaginations are long and convoluted producing, in section, numerous irregular profiles (Fig. 4). They carry with them extensions of the underlying basal famina which, in this region of the foregut, is wide (approx. 0.4 pm) and extends around the pharynx as a skeletal framework for muscle attachment.Hemidesmosomes provide insertion points for the numerous radial and circular muscles that make up the bulk of the pharynx (Fig. 4). Mitochondria generally occupy a basal position in the cytoplasm where they are closely associated with the invag~nations of basal membrane. They are irregular in shape and have relatively few cristae and very pale matrices. In places the epithefium is occupied by clusters of dense, membrane-bound inclusions of varying size and shape and containing stacks or concentric arrays of membrane (Fig. 3, inset). Their origin or function is unknown. Nuclei were never observed in the epithelium nor were connections to subjacent perikarya. Junctions between the pharynx epithelium and tegument of the mouth cavity have not been seen so it is possible that the two structures are confluent. The pharyngeaf fining extends for a short distance beyond the main body of the pharynx before being replaced by the oesophageaf lining. At their junction the two epithelia are connected by an apical septate desmosome (Fig. 5). Oesophtrgrrs
The oesphagus is short and measures approx. 100-200 I_tm in length. It is fined by an irregularly folded, syncytiaf epithelium (Fig. 6). Invaginations of the plasma membrane are common and penetrate deep into the cytoplasm. In common with the pharyngeal epithelium, the infolded membranes of the oesphagus are in close apposition and appear as multilaminate structures. Branched indense, vaginations of the basal membrane are extensive and carry with them material that is continuous with the underlying basal lamina. The basal lamina in this region is generally much thinner than that of the pharynx, measuring approx. 0.1-0.2 pm. A muscular coat of inner longitudinal and outer circular muscle fibres surrounds the oesophagus
Foregut
I.J.P. VOL. 6. 1976
FIG. I. Section
through
the mouth
and glands
tegument
of Cu/icory/e
519
showing
the characteristic secretory inclusion bodies infoldings of surface membrane (*) and slot-like imaginations of basal membrane (unlabelled arrows), and the supporting fibrillar tissues: basal lamina (BL), fibrous interstitial material (IM) and muscle (Mu). Inset shows detail of the two secretory inclusion bodies of the tegument. The spherical form has a granular content and appears to liberate its contents into the cytoplasm (arrow). Note the dense, subsurface layer of cytoplasm (*) and the external fuzzy coat.
in the form of dense spheres (SD) and rods (RS). Note the pit-like
FIG. 2. Section through a tegumentary cell body containing developing spherical-type secretory inclusions (*). Their formation involves the GER and Golgi stacks (Go). Nu, nucleus; Par, parenchyma.
and is continuous with the musculature of the two caeca. Nuclei were not observed in the oesophageal epithelium and evidence of perikarya or of cytoplasmic connections to the epithelium was not forthcoming. The bulk of the cytoplasm of the epithelium is filled with membrane-limited vesicular
inclusions that vary greatly in size and shape and contain a scant fibrous or granular substance (Fig. 5). Their origin has not been determined. Oval and elongate mitochondria with relatively few cristae and pale matrices are present and, as in the pharyngeal epithelium, are most numerous in the basal cytoplasm and close to the invaginations of basal
520
D. W. HALTON and S. D.
STKANOCK
I.J.P. VOL. 6. 1976
FIG. 3. Transverse section through part of the pharynx showing the triradiate lumen (Lu) epithelial lining. Note the numerous, dense invaginations of surface plasma membrane (In) pharynx muscles (Mu). Inset shows detail of the membranous inclusions that characterize epithelium.
and and the
FIG. 4. The deep invaginations of surface membrane (arrows) of the pharynx epithelium are matched by the long and often convoluted infoldings of basal membrane which produce, in section, numerous irregular profiles (BI). Note the numerous hemidesmosomes (HD) which anchor the circular and radial muscle fibres to the extensive basal lamin (*) Lu, lumen. membl -ane. Elements of an endoplasmic reticulum or Go1 gi apparatus are apparently absent. to the bifurcation of the gut the Just anterior oesopk lageal epithelium is replaced by the caecal epithel ium, and the point of union is marked by a long se :pta.te desmosome (Fig. 7).
Oesophuged glut7A The oesophageal glands are situated in the chyma on either side of the oesophagus and from a point in line with the mid-region pharynx to one just posterior to the bifurca the gut. They are unicellular and have cytol
parenextend of the tion of 3lasmic
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FIG.
1976
Foregut
5. Section
through
and glands of Cnlicot~~le
the septate desmosome (Ph) and oesophageal
(arrow) which marks the junction (Oe) epithelia. Lu, lumen.
of the pharyngeal
FIG. 6. Transverse section through part of the oesophagus epithelium showing the deep imaginations of plasma membrane (arrows) and secretion-filled ducts which open to the lumen (Lu). Note the basal lamina (*) and associated musculature (Mu). FK.
7. The
oesophageal
epithelium
(Oe)
is connected
septate desmosome extensions connecting to the oesophageal lumen. Both the cells and the extensions are invested with a thin layer of fibres that form part of a network of interstitial material supporting the various organ systems in the worm. The glands are irregular in outline with invaginations of plasma membrane and associated interstitial material penetrating deep
to the caecal
epithelium
(CE)
by a
long
(arrow).
into the cytoplasm (Fig. 8). Many of the larger invaginations of plasma membrane also contain extensions of adjacent parenchymal cells and, in localized areas, the limiting membranes of the two cell types come together in the form of a close junction (Fig. IO). Each
gland
cell
has a large
central
nucleus
con-
D. W. HALT~N and S. D. STRAMKX
FIG. 8. Section through dense secretory droplets
FIG. 9. Detail droplets (SD).
I.J.P. VOL. 6. 1976
two oesophageal gland cells showing the nucleus (Nuf, extensive GER (SD). Note the irregular outline of the cells and the deep inserkns interstitial fibres (“1 and parenchyma (Par).
and ot
of the structural relationship between the GER, Golgi apparatus (Go) and secretory Note nucleus (Nu) and ports in nuclear envelope (arrows) which, in places (*), is in continuity with GER.
mining finely granular nucleoplasm and small areas of condensed chromatin (Fig. 9). The nucleolus is composed of a fibrous portion and of regions of dense granules that resemble cytoplasmic ribosomes. Pores occur at intervals in the nuclear envelope, and there is some densitkation of the associated nucleoplasm and cytoplasm. The nuclear
envelope is, in places, in continuity with cisternae of rough-surfaced ER, and its outer component is encrusted with ribosomes. Free ribosomes account for much of the ground substance of the cell. Mitochondria are distributed in all regions of the cell and are either round or oval in profile with pale matrices and comparatively few, short cristae.
Foregut
I.J.P. VOL. 6. 1976
Fw.
IO. Section
FIG. 11. Section secretory droplet
FIG.
through
and glands
523
of Calicotyle
a close junction (arrow) between the limiting membranes gland cell (OG) and parenchymal cell (Par).
of an oesophageal
through the Golgi region (Go) of an oesophageal gland showing an immature (*) which has been formed by fusion of Golgi-derived vesicles. The secretion has undergone some condensation in the more mature droplets (SD).
12. Section through part of an oesophageal gland duct where it penetrates and basal lamina (BL) of the oesophagus. Note the microtubular lining (arrow)
FIG. 13. Section through the terminal region of a gland cell duct. Note that desmosome (**) anchors the duct to the oesophageal epithelium. Arrow indicates
the muscles (Mu) in this region. an annular oesophagus
septate lumen.
524
D. W. HALT~N and S. D. STRANO~K
The bulk of the cytoplasm of each gland is filled with long, narrow cisternae of GER. Golgi stacks are numerous and comprise several flattened saccules with a dense content (Fig. I I). The forming face of the stack is structurally related to the ER, while the more distal saccules often terminate in small, bulbous swellings (Figs. 9 & I I). These give rise by abscission to Golgi vesicles that coalesce to form the immature secretory droplets (Fig. 9). The droplets are membrane-bound and contain a moderately-dense substance that undergoes condensation with development. When fully formed the droplets measure approx. 0.5-0.6 pm in diameter and occupy most of the duct-like extensions of the cell, together with occasional mitochondria and small profiles of ER. As the ducts approach the oesophagus they become closely packed, being separated only by thin segments of parenchymal tissue and interstitial fibres. At this point the ducts are lined by microtubules which are closely-spaced at regular intervals just below the limiting membrane. This microtubular lining is particularly evident where the ducts penetrate the muscles of the oesophagus and enter the oesophageal epithelium (Fig. 12). An annular septate desmosome connects the plasma membrane of the terminal part of the duct to that of the epithelium (Fig. 13). The duct termini are often found swollen with secretory droplets, but their release to the oesophagus lumen was not observed. DISCUSSION This study has shown that, although there is not the same complexity of structure or multiplicity of glands, the anterior alimentary tract of the monopisthocotylean, Calicoryle is morphologically similar to that of the polyopisthocotylean, Diclidophora. In both monogeneans an invagination of external tegument lines the oral or buccal cavity and there are distinct epithelia lining the pharynx and oesophagus, with numerous gland cells opening into the lumen. The tegument of the mouth cavity of Calicotyle shows no distinctive features, being similar in appearance to that covering the general body surface. The same two types of tegumentary inclusion body can be found in most areas of tegument and there is a well developed filamentous coat covering the entire body surface. Regional differences have been previously found, however, in the distribution of histochemically demonstrable carboxylic esterase. Halton & Jennings (1965) showed that reactivity for the enzyme was localized in the tegument of the anterior ventral region, as well as in that lining the oral sucker. Inhibitor studies (Halton, unpublished data) have shown the esterase to be a specific acetylcholinesterase similar to that which characterizes cholinergic components of the vertebrate neuromuscular system. The role of this enzyme in
I.J.P. VOL.
6.
1976
areas of tegument that are in contact with the cloaca1 or rectal wall of the fish could be to facilitate attachment and feeding of the parasite by minimising the level of muscular activity in the host tissues at these sites. Sense organs have not been observed in the mouth tegument and are probably few in number or absent In contrast, the same region in Diclidophorn is marked by the presence of numerous uniciliate receptors with a presumed tactile function (Halton & Morris, 1975). Similarly, there are very few glands associated with the mouth in Culicotyk, in contrast to Diclidophorn (Halton, Morris & Hardcastle. 1974), and dense, spiny areas of tegument, such as distinguish the mouth tegument of Diclidophorcr, are absent from Cnlicotyle. These species differences in foregut morphology would seem to reflect not only the respective systematic positions of the two worms but also the differences in their diet and digestive mechanisms. Cnlicotyle browses on the cloaca1 or rectal lining of the host fish, ingesting mucus and desquamated epidermal cells. Attachment of the anterior end of the worm is presumably effected by the oral sucker, wlith adhesive (sticky) glands being unnecessary. Similarly, histolytic glands or an abrasive, spined tegument would also seem to be unnecessary since food is directly available for ingestion. It is of interest to note that Halton & Jennings (1965) found no evidence of damage to the cloaca1 wall and its epidermis as a result of feeding, suggesting the pharynx does not remove living epidermal cell or breach the epidermis. Diclidopharn, on the other hand, is sanguinivorous and has to find and breach a blood vessel in order to ingest blood, and, at the same time, secure the oral region to the feeding site for the duration of the meal. For these reasons, the sense receptors, spined tegument, and histolytici adhesive/anticoagulant secretions that can be found in the foregut of the gill fluke have obvious significance. The pharynx of Cnlicotyle is a well defined, highly muscular structure devoid of glands. It functions as a suctorial feeding organ in what appears to be a purely mechanical feeding process (Halton & Jennings, 1965). The present ultrastructural findings do not conflict with this view. The basal lamina, which supports both the tegument and internal epithelial linings of the gut, is particularly well developed in the pharynx. It provides for an increase in strength in this region by forming an extensive framework for muscle attachment, the sites of which are marked by numerous hemidesmosomes. Moreover, its insertion into the overlying epithelium undoubtedly furnish support and anchorage to the lining of the pharynx. This would seem to be necessary in view of the stresses imposed on the structure by the activity of the pharyngeal musculature. The deep surface infoldings which characterize the epithelium of the pharynx
I.J.P. VOL. 6.
I976
Foregut and glands of Culicotyle
in contracted state also indicate that the lining can accommodate luminal distension and compression resulting from the pulsatory action of the pharynx during feeding. In contrast to the condition found in Diclidophora, the morphology of the pharyngeal epithelium of Calicotyle is not consistent with a structure involved in absorption. Amplification of the luminal surface area by microvilli or lamellar outfoldings was not evident. Again, this would seem to reflect the adaptive features shown by the two worms to different diets, with blood presenting far more low molecular weight compounds for direct absorption in the foregut of Diclidophora than mucus and desquamated epithelial cells must do in Calicotyle. Again, this points to the pharynx of Calicotyle functioning solely as a mechanical aid to feeding, emphasising the greater need for an efficient muscular organ when sucking in mucus and associated epidermal cells, as distinct from a semi-fluid food such as blood. The secretory elements of the anterior alimentary tract of Calicotyle occur as extensive unicellular glands opening via long duct-like structures into the oesophagus. The glands are morphologically reminiscent of protein-secreting cells, containing dense secretory droplets whose formation involves the GER and Golgi apparatus in the production of proteins for export. The protein components are synthesized by the ribosomes of the GER and transferred to the Golgi apparatus for packaging and concentration as membrane-bound droplets. The droplets migrate along the duct-like extensions of the cells and are periodically discharged into the oesophagus lumen. They are acidophilic and stain positively for proteins, using the mercuric bromphenol blue test, and it is assumed that they contain the enzymes necessary for the initial extracellular phase of digestion. The glands form a close spatial relationship with the surrounding parenchyma, deep insertions of which penetrate the cell and form close junctions between the plasma membranes of the two cell types. In this way the nucleus and GER cisternae of the gland are brought into close association with the parenchyma whose food storage and circulatory capacity means that a supply of precursor molecules will be readily available for the synthesis of RNA and protein secretion. The preponderance of GER and Golgi stacks in the cytoplasm is indicative of an active and perhaps continuous production of and since the glands most probably secretion, function for the life of the worm a constant supply of precursor molecules would seem to be of paramount importance. The duct-like extensions of the glands have a microtubular lining in the region where they penetrate the muscle layers, fibrous sheath and epithelium of the oesophagus. It is assumed that the tubules, together with the terminal septate desmosome,
525
strengthen and support the ducts in a region that is subjected to the muscular contractions of the feeding apparatus, and so allow a free flow of secretion to the gut lumen. The mechanism by which the secretion is discharged is not known, but the absence of intact secretory droplets in the oseophagus lumen suggests it is a merocrine type, involving membrane fusion. The actual release may be triggered by distension of the oesophagus lumen, such as would occur with the influx of food, so ensuring synchrony in the mixing of enzyme and substrate. Gland cells appear to be a distinguishing feature of the foregut of monogeneans. They have been recorded in light microscopic descriptions of other monopisthocotyleans, such as Entobdella solear and Acanthocotyle sp. by Kearn (1963) and E. hippoglossi by Halton & Jennings (1965) as well as for polyopisthocotyleans like Polystoma integerrimum by Williams (I 96l), Polystomoides sp. by Rohde (1974) and the gill flukes, Diclidophora merlangi, D. denticulata, Diplozoom paradoxum, Discocotyle sagittata and Plectanocotyle gurnardi by Halton & Jennings (1965) and Halton (1974). On the other hand, the anterior part of the alimentary tract of digenetic trematodes is generally nonglandular, with an extension of the body tegument lining not only the oral sucker but also the pharynx and oesophagus. In these worms, in contrast to the monogeneans so far examined by electron microscopy (Halton, 1975; Halton & Stranock, 1976), the cells of the caecal epithelium are both secretory and absorptive in function and provide the necessary hydrolytic enzymes for the extracellular phase of digestion (Halton, 1967; Howell, 1973 ; Robinson & Threadgold, 1975). The so-called oesophageal gland of Schistosoma, which appears to function in the intraluminar digestion of ingested blood cells, is in fact a modified form of oesophageal tegument, rather than a separate collection of gland cells (Morris & Threadgold. 1968; Spence & Silk, 1970; Dike, I97 I ). Achtro~ledgemrnl-apart of this work was undertaken while S.D.S. was in receipt of a Science Research Council Postgraduate Studentship.
REFERENCES S. C. 1971. Ultrastructure region in Schistosoma mansoni.
DIKE
Tropical
Medicine
and Hygiene
of the oesophageal American Journal 20: 552-568.
of
D. W. 1967. Observations on the nutrition of digenetic trematodes. Parasitology 57: 639-660. HALTON D. W. 1974. Functional aspects of monogenean gut cells. Proceedings of the 3rd Internariorlal Congrexv ofParasitology, Munich, August 1974, pp. 415-416. HALTON D. W. 1975. Intracellular digestion and cellular defecation in a monogenean, Diclidophora merlangi. HALTON
Parasitology
70: 331-340.
526
D. W. HALTON and S. D. STRANO~K
HALTON D. W. & JENNINGS J. B. 1965. Observations on the nutrition of monogenetic trematodes. Bio/ogicuI Bulletin, Woods Hole, Massachusetts 129: 257-272. HALTON D. W., MORRIS G. P. & HARDCASTLE A. 1974. Gland cells associated with the alimentary tract of a monogenean, Diclidophora merlungi. Internationul Journal for Parasitology 4: 489-599. HALTON D. W. & MORRIS G. P. 1975. Ultrastructure of the anterior alimentary tract of a monogenean, Diclidophora merlungi. Internationul Journal for Parasifology 5 : 4074 19. HALTON D. W. & STRANOCK S. D. 1976. The line structure and histochemistry of the caecal epithelium of Calicoryle ltr6yeri (Monogenea: Monopisthocotylea). International Journal for Partritology 6: 253-263. HOWELL M. J. 1973. Localization of protcolytic activity in Fasciola hepatiea. The Journul c1.f’Pararitology 59: 454-456. KEARN C. C. 1963. Feeding in some monogencan skin parasites: Entobdella soleae on Solecr solea and Acanthocotyle sp. on Raia clavata. Journal of the Marine
1.J.P. VOL. 6. 1976
Biological Association of the United Kingdom 43: 749-766. MORRIS G. P. & THKEADGOLD L. T. 1968. Ultrastructure of the tegument of the adult Schistosoma mansoni. The Journal of Parasitology 54 : I 5-27. ROBINSON G. & THREADGOLD L. T. 1975. Electron microscope studies of Fasciola hepatica. XII. The fine structure of the gastrodermis. Experimental Parasitology 37: 20-36. ROI~DE K. 1974. Light and electron-microscopic studies of the pharynx and the anterior and posterior glands of Polystomoides (Monogenea: Folystomatidae). Zoologische Jahrbiicher Ahtreilung fiir Anatomie 92: l-17. SPENCE 1. M. & SILK M. H. 1970. Ultrastructural studies of the blood fluke~Schistosoma mansoni. IV. The digestive system. South African Journal of Medical Sciences 35: 93-112. WILLIAMS J. B. 1961. The dimorphism of Polystoma integerrimum (Friilich) Rudolphi and its bearing on relationships within the Polystomatidae: Part I. Journal of Helminthology 34: 151-192.
OF THE FOREGUT AND ASSOCIATED
GLANDS
OF CALZCOTYLE
KRGYERZ
(MONOGENEA : MONOPISTHOCOTYLEA) D. W. HALTON and S. D. STRANOCK Department
of Zoology,
The Queen’s (Received
University,
Belfast
28 Junuary
BT7 1NN,
Northern
Ireland
1976)
Abstract--HALToN D. W. and STRANOCK S. D. 1976. Ultrastructure of the foregut and associated glands of Calicoryle krGyeri (Monogenea: Monopisthocotylea). International Journal fur Parasitology 6: 517526. The mouth, pharynx and oesophagus of Calicotyle are lined by syncytial epithelia, and there are numerous unicellular glands associated with the oesophagus. An infolding of unmodified external tegument lines the mouth cavity and is connected by discrete cytoplasmic processes to subjacent perikarya. It contains two types of secretory body and its luminal surface is invested with a finely filamentous coating. The pharynx and oesophagus are lined by irregularly-folded epithelia that are interconnected by a septate desmosome. Membranous inclusions distinguish the pharynx epithelium and there is a w-ell developed basal lamina for insertion of the pharyngeal muscles. The oesophagus epithelium is perforated by the openings of the oesophageal glands. These lie in the surrounding parenchyma and produce a dense, membrane-bound secretion which is conveyed by duct-like extensions of the glands to the oesophagus lumen. The ducts are supported in places by microtubules and are anchored to the oesophageal epithelium by septate desmosomes. A septate desmosome also marks the junction between the epithelium and the gut caeca.
INDEX KEY WORDS: Calicotyle alimentary tract; gland cells.
krdveri;
monogenean;
microscopy,
INTRODUCTION
The
anterior part of the alimentary tract of consists of a mouth (surrounded by an oral sucker), pharynx and oesophagus, each of which is lined by a separate epithelium. An extensive collection of unicellular glands are situated on either side of the oesophagus and open into its lumen. Immediately behind the gland cell openings the gut divides into the two blind-ending intestinal caeca. C. kriiyeri
Mouth
cavity
The mouth is lined by tegument which is continuous with that covering the external body surface. Its general organization corresponds to that found in other parasitic platyhelminthes in that there are nucleated secretory regions--tegumentary cell bodies or perikarya-positioned deep in the parenchyma which connect by discrete cytoplasmic processes to an anucleate surface syncytium. This outer syncytial layer in Crrlicotyle is approx. 1.5-2 pm in thickness and is penetrated at fairly regular intervals by a series of surface infoldings or pits (Fig. 1). The pits are generally quite narrow in diameter and measure approx. 0.4 pm in depth. Morphological evidence of endocytosis, such as coated invaginations and related vesicles, was not observed. The plasmalemma of the tegument is asymmetric and displays apparent
digestion.
The work forms part of a comparative electron microscope study of gut structure in skin-feeding monopisthocotylean and blood-feeding polyopisthocotylean monogeneans which, it is hoped, will form a base-line for physiological investigation of nutrition in these parasites. AND
ultrastructure;
RESULTS
Cdicotyle kriiyeri inhabits the cloaca and rectum of skate and ray and feeds on desquamated epidermal cells and mucus. The food is digested by a combination of extraand intracellular processes, involving oesophageal glands and the caecal epithelium (Halton & Jennings, 1965). Ultrastructural studies by Halton & Stranock (1976) have already shown the caecal cells to be involved in endocytosis and intracellular digestion of mucus and epidermal cell debris. The present observations on the foregut and associated glands expand these studies to include morphological detail of the elements responsible for the extracellular phase of
MATERIALS
electron;
METHODS
Details of the collection of specimens of C. krtiyeri and their preparation for examination by electron microscopy have been given elsewhere (Halton & Stranock, 1976). 517
518
D.W.
HALTON~~~S.D.STRANOCK
continuity with a glycocalyx-like, filamentous coat (Fig. I, inset). Beneath the membrane there is a dense subsurface layer of finely fibrous material below which the tegument matrix is less dense and contains mitochondria and numerous dense secretory bodies. The mitochondria are oval or elongate in outline with moderately-dense matrices and relatively few transverse cristae. Although present in most parts of the tegumentary unit, the majority of mitochondria are concentrated near the basal plasma membrane. Two types of secretory inclusion body can be recognized in the tegument. The most numerous are membrane-bound spheres measuring between 0.15 and 0.3 pm in diameter and displaying a packed granular content (Fig. 1). They are present in all areas of the tegument and are formed in the subjacent ceil bodies. A number of them display increased granulation of content in the surface tegument and, in some instances, appear to release their content to the cytoplasm (Fig. 1, inset). The other inclusion bodies are rod-shaped and contain a central core of dense material (Fig. I). They measure approx. 0.1 pm in diameter and vary in length, appearing either straight or slightly curved. Their origin is unclear since they have not been observed in any of the tegumentary cell bodies examined. The cell bodies can be identified by the presence in their cytoplasm of the spherical tegumentary inclusion bodies in various stages of development (Fig. 2). The nucleus is large and ovoid and there are long narrow profiles of GER and numerous Golgi stacks involved in the formation of the secretion. The plasma membrane is irregular in outline and close junctions with adjacent parenchyma1 cells are fairly common. The basal plasma membrane of the surface tegument is frequently folded, forming short, slotlike invaginations of fairly constant width (30 nm) (Fig. 1). They occur singly or in groups of three or four in parallel array. Larger invaginations of variable width are also present and contain material that is continuous with the underlying basal lamina. These membranous configurations are usually branched and often extend as far as the apical cytoplasm. The basal lamina comprises a layer (approx. 0.1 pm in thickness) of finely fibrous material underneath the basal plasma membrane but separated from it by a narrow gap (Fig. 1). Below this, thick fibrous interstitial material fills all the available space between the tegumentary unit and the muscle fibres of the oral sucker. In places, this sheath of fibrous material is penetrated by spike-like insertions of the basal lamina. Pharynx
The pharynx is a large, well-developed muscular structure measuring approx. 450 pm in length and
1.J.P. VOL.6.1976
350 wrn maximum width. Its lumen is triradiate and fined by an apparently syncytiaf epithelium, measuring some 1.53 pm in height (Fig. 3). In all the specimens examined, the pharynx appears constricted so that its fining is highly folded, with undulant and frequently branched surface invaginations penetrating deep into the cytoplasm. The free surfaces of the infolded membrane are closely apposed producing a dense, multilaminate appearance in section (Figs. 3 & 4). The basal plasma membrane is also infolded, with broad finger-like invaginations of membrane extending in curved profiles for some distance into the cytoplasm. In places the invaginations are long and convoluted producing, in section, numerous irregular profiles (Fig. 4). They carry with them extensions of the underlying basal famina which, in this region of the foregut, is wide (approx. 0.4 pm) and extends around the pharynx as a skeletal framework for muscle attachment.Hemidesmosomes provide insertion points for the numerous radial and circular muscles that make up the bulk of the pharynx (Fig. 4). Mitochondria generally occupy a basal position in the cytoplasm where they are closely associated with the invag~nations of basal membrane. They are irregular in shape and have relatively few cristae and very pale matrices. In places the epithefium is occupied by clusters of dense, membrane-bound inclusions of varying size and shape and containing stacks or concentric arrays of membrane (Fig. 3, inset). Their origin or function is unknown. Nuclei were never observed in the epithelium nor were connections to subjacent perikarya. Junctions between the pharynx epithelium and tegument of the mouth cavity have not been seen so it is possible that the two structures are confluent. The pharyngeaf fining extends for a short distance beyond the main body of the pharynx before being replaced by the oesophageaf lining. At their junction the two epithelia are connected by an apical septate desmosome (Fig. 5). Oesophtrgrrs
The oesphagus is short and measures approx. 100-200 I_tm in length. It is fined by an irregularly folded, syncytiaf epithelium (Fig. 6). Invaginations of the plasma membrane are common and penetrate deep into the cytoplasm. In common with the pharyngeal epithelium, the infolded membranes of the oesphagus are in close apposition and appear as multilaminate structures. Branched indense, vaginations of the basal membrane are extensive and carry with them material that is continuous with the underlying basal lamina. The basal lamina in this region is generally much thinner than that of the pharynx, measuring approx. 0.1-0.2 pm. A muscular coat of inner longitudinal and outer circular muscle fibres surrounds the oesophagus
Foregut
I.J.P. VOL. 6. 1976
FIG. I. Section
through
the mouth
and glands
tegument
of Cu/icory/e
519
showing
the characteristic secretory inclusion bodies infoldings of surface membrane (*) and slot-like imaginations of basal membrane (unlabelled arrows), and the supporting fibrillar tissues: basal lamina (BL), fibrous interstitial material (IM) and muscle (Mu). Inset shows detail of the two secretory inclusion bodies of the tegument. The spherical form has a granular content and appears to liberate its contents into the cytoplasm (arrow). Note the dense, subsurface layer of cytoplasm (*) and the external fuzzy coat.
in the form of dense spheres (SD) and rods (RS). Note the pit-like
FIG. 2. Section through a tegumentary cell body containing developing spherical-type secretory inclusions (*). Their formation involves the GER and Golgi stacks (Go). Nu, nucleus; Par, parenchyma.
and is continuous with the musculature of the two caeca. Nuclei were not observed in the oesophageal epithelium and evidence of perikarya or of cytoplasmic connections to the epithelium was not forthcoming. The bulk of the cytoplasm of the epithelium is filled with membrane-limited vesicular
inclusions that vary greatly in size and shape and contain a scant fibrous or granular substance (Fig. 5). Their origin has not been determined. Oval and elongate mitochondria with relatively few cristae and pale matrices are present and, as in the pharyngeal epithelium, are most numerous in the basal cytoplasm and close to the invaginations of basal
520
D. W. HALTON and S. D.
STKANOCK
I.J.P. VOL. 6. 1976
FIG. 3. Transverse section through part of the pharynx showing the triradiate lumen (Lu) epithelial lining. Note the numerous, dense invaginations of surface plasma membrane (In) pharynx muscles (Mu). Inset shows detail of the membranous inclusions that characterize epithelium.
and and the
FIG. 4. The deep invaginations of surface membrane (arrows) of the pharynx epithelium are matched by the long and often convoluted infoldings of basal membrane which produce, in section, numerous irregular profiles (BI). Note the numerous hemidesmosomes (HD) which anchor the circular and radial muscle fibres to the extensive basal lamin (*) Lu, lumen. membl -ane. Elements of an endoplasmic reticulum or Go1 gi apparatus are apparently absent. to the bifurcation of the gut the Just anterior oesopk lageal epithelium is replaced by the caecal epithel ium, and the point of union is marked by a long se :pta.te desmosome (Fig. 7).
Oesophuged glut7A The oesophageal glands are situated in the chyma on either side of the oesophagus and from a point in line with the mid-region pharynx to one just posterior to the bifurca the gut. They are unicellular and have cytol
parenextend of the tion of 3lasmic
I.J.P.
VOL.
6.
FIG.
1976
Foregut
5. Section
through
and glands of Cnlicot~~le
the septate desmosome (Ph) and oesophageal
(arrow) which marks the junction (Oe) epithelia. Lu, lumen.
of the pharyngeal
FIG. 6. Transverse section through part of the oesophagus epithelium showing the deep imaginations of plasma membrane (arrows) and secretion-filled ducts which open to the lumen (Lu). Note the basal lamina (*) and associated musculature (Mu). FK.
7. The
oesophageal
epithelium
(Oe)
is connected
septate desmosome extensions connecting to the oesophageal lumen. Both the cells and the extensions are invested with a thin layer of fibres that form part of a network of interstitial material supporting the various organ systems in the worm. The glands are irregular in outline with invaginations of plasma membrane and associated interstitial material penetrating deep
to the caecal
epithelium
(CE)
by a
long
(arrow).
into the cytoplasm (Fig. 8). Many of the larger invaginations of plasma membrane also contain extensions of adjacent parenchymal cells and, in localized areas, the limiting membranes of the two cell types come together in the form of a close junction (Fig. IO). Each
gland
cell
has a large
central
nucleus
con-
D. W. HALT~N and S. D. STRAMKX
FIG. 8. Section through dense secretory droplets
FIG. 9. Detail droplets (SD).
I.J.P. VOL. 6. 1976
two oesophageal gland cells showing the nucleus (Nuf, extensive GER (SD). Note the irregular outline of the cells and the deep inserkns interstitial fibres (“1 and parenchyma (Par).
and ot
of the structural relationship between the GER, Golgi apparatus (Go) and secretory Note nucleus (Nu) and ports in nuclear envelope (arrows) which, in places (*), is in continuity with GER.
mining finely granular nucleoplasm and small areas of condensed chromatin (Fig. 9). The nucleolus is composed of a fibrous portion and of regions of dense granules that resemble cytoplasmic ribosomes. Pores occur at intervals in the nuclear envelope, and there is some densitkation of the associated nucleoplasm and cytoplasm. The nuclear
envelope is, in places, in continuity with cisternae of rough-surfaced ER, and its outer component is encrusted with ribosomes. Free ribosomes account for much of the ground substance of the cell. Mitochondria are distributed in all regions of the cell and are either round or oval in profile with pale matrices and comparatively few, short cristae.
Foregut
I.J.P. VOL. 6. 1976
Fw.
IO. Section
FIG. 11. Section secretory droplet
FIG.
through
and glands
523
of Calicotyle
a close junction (arrow) between the limiting membranes gland cell (OG) and parenchymal cell (Par).
of an oesophageal
through the Golgi region (Go) of an oesophageal gland showing an immature (*) which has been formed by fusion of Golgi-derived vesicles. The secretion has undergone some condensation in the more mature droplets (SD).
12. Section through part of an oesophageal gland duct where it penetrates and basal lamina (BL) of the oesophagus. Note the microtubular lining (arrow)
FIG. 13. Section through the terminal region of a gland cell duct. Note that desmosome (**) anchors the duct to the oesophageal epithelium. Arrow indicates
the muscles (Mu) in this region. an annular oesophagus
septate lumen.
524
D. W. HALT~N and S. D. STRANO~K
The bulk of the cytoplasm of each gland is filled with long, narrow cisternae of GER. Golgi stacks are numerous and comprise several flattened saccules with a dense content (Fig. I I). The forming face of the stack is structurally related to the ER, while the more distal saccules often terminate in small, bulbous swellings (Figs. 9 & I I). These give rise by abscission to Golgi vesicles that coalesce to form the immature secretory droplets (Fig. 9). The droplets are membrane-bound and contain a moderately-dense substance that undergoes condensation with development. When fully formed the droplets measure approx. 0.5-0.6 pm in diameter and occupy most of the duct-like extensions of the cell, together with occasional mitochondria and small profiles of ER. As the ducts approach the oesophagus they become closely packed, being separated only by thin segments of parenchymal tissue and interstitial fibres. At this point the ducts are lined by microtubules which are closely-spaced at regular intervals just below the limiting membrane. This microtubular lining is particularly evident where the ducts penetrate the muscles of the oesophagus and enter the oesophageal epithelium (Fig. 12). An annular septate desmosome connects the plasma membrane of the terminal part of the duct to that of the epithelium (Fig. 13). The duct termini are often found swollen with secretory droplets, but their release to the oesophagus lumen was not observed. DISCUSSION This study has shown that, although there is not the same complexity of structure or multiplicity of glands, the anterior alimentary tract of the monopisthocotylean, Calicoryle is morphologically similar to that of the polyopisthocotylean, Diclidophora. In both monogeneans an invagination of external tegument lines the oral or buccal cavity and there are distinct epithelia lining the pharynx and oesophagus, with numerous gland cells opening into the lumen. The tegument of the mouth cavity of Calicotyle shows no distinctive features, being similar in appearance to that covering the general body surface. The same two types of tegumentary inclusion body can be found in most areas of tegument and there is a well developed filamentous coat covering the entire body surface. Regional differences have been previously found, however, in the distribution of histochemically demonstrable carboxylic esterase. Halton & Jennings (1965) showed that reactivity for the enzyme was localized in the tegument of the anterior ventral region, as well as in that lining the oral sucker. Inhibitor studies (Halton, unpublished data) have shown the esterase to be a specific acetylcholinesterase similar to that which characterizes cholinergic components of the vertebrate neuromuscular system. The role of this enzyme in
I.J.P. VOL.
6.
1976
areas of tegument that are in contact with the cloaca1 or rectal wall of the fish could be to facilitate attachment and feeding of the parasite by minimising the level of muscular activity in the host tissues at these sites. Sense organs have not been observed in the mouth tegument and are probably few in number or absent In contrast, the same region in Diclidophorn is marked by the presence of numerous uniciliate receptors with a presumed tactile function (Halton & Morris, 1975). Similarly, there are very few glands associated with the mouth in Culicotyk, in contrast to Diclidophorn (Halton, Morris & Hardcastle. 1974), and dense, spiny areas of tegument, such as distinguish the mouth tegument of Diclidophorcr, are absent from Cnlicotyle. These species differences in foregut morphology would seem to reflect not only the respective systematic positions of the two worms but also the differences in their diet and digestive mechanisms. Cnlicotyle browses on the cloaca1 or rectal lining of the host fish, ingesting mucus and desquamated epidermal cells. Attachment of the anterior end of the worm is presumably effected by the oral sucker, wlith adhesive (sticky) glands being unnecessary. Similarly, histolytic glands or an abrasive, spined tegument would also seem to be unnecessary since food is directly available for ingestion. It is of interest to note that Halton & Jennings (1965) found no evidence of damage to the cloaca1 wall and its epidermis as a result of feeding, suggesting the pharynx does not remove living epidermal cell or breach the epidermis. Diclidopharn, on the other hand, is sanguinivorous and has to find and breach a blood vessel in order to ingest blood, and, at the same time, secure the oral region to the feeding site for the duration of the meal. For these reasons, the sense receptors, spined tegument, and histolytici adhesive/anticoagulant secretions that can be found in the foregut of the gill fluke have obvious significance. The pharynx of Cnlicotyle is a well defined, highly muscular structure devoid of glands. It functions as a suctorial feeding organ in what appears to be a purely mechanical feeding process (Halton & Jennings, 1965). The present ultrastructural findings do not conflict with this view. The basal lamina, which supports both the tegument and internal epithelial linings of the gut, is particularly well developed in the pharynx. It provides for an increase in strength in this region by forming an extensive framework for muscle attachment, the sites of which are marked by numerous hemidesmosomes. Moreover, its insertion into the overlying epithelium undoubtedly furnish support and anchorage to the lining of the pharynx. This would seem to be necessary in view of the stresses imposed on the structure by the activity of the pharyngeal musculature. The deep surface infoldings which characterize the epithelium of the pharynx
I.J.P. VOL. 6.
I976
Foregut and glands of Culicotyle
in contracted state also indicate that the lining can accommodate luminal distension and compression resulting from the pulsatory action of the pharynx during feeding. In contrast to the condition found in Diclidophora, the morphology of the pharyngeal epithelium of Calicotyle is not consistent with a structure involved in absorption. Amplification of the luminal surface area by microvilli or lamellar outfoldings was not evident. Again, this would seem to reflect the adaptive features shown by the two worms to different diets, with blood presenting far more low molecular weight compounds for direct absorption in the foregut of Diclidophora than mucus and desquamated epithelial cells must do in Calicotyle. Again, this points to the pharynx of Calicotyle functioning solely as a mechanical aid to feeding, emphasising the greater need for an efficient muscular organ when sucking in mucus and associated epidermal cells, as distinct from a semi-fluid food such as blood. The secretory elements of the anterior alimentary tract of Calicotyle occur as extensive unicellular glands opening via long duct-like structures into the oesophagus. The glands are morphologically reminiscent of protein-secreting cells, containing dense secretory droplets whose formation involves the GER and Golgi apparatus in the production of proteins for export. The protein components are synthesized by the ribosomes of the GER and transferred to the Golgi apparatus for packaging and concentration as membrane-bound droplets. The droplets migrate along the duct-like extensions of the cells and are periodically discharged into the oesophagus lumen. They are acidophilic and stain positively for proteins, using the mercuric bromphenol blue test, and it is assumed that they contain the enzymes necessary for the initial extracellular phase of digestion. The glands form a close spatial relationship with the surrounding parenchyma, deep insertions of which penetrate the cell and form close junctions between the plasma membranes of the two cell types. In this way the nucleus and GER cisternae of the gland are brought into close association with the parenchyma whose food storage and circulatory capacity means that a supply of precursor molecules will be readily available for the synthesis of RNA and protein secretion. The preponderance of GER and Golgi stacks in the cytoplasm is indicative of an active and perhaps continuous production of and since the glands most probably secretion, function for the life of the worm a constant supply of precursor molecules would seem to be of paramount importance. The duct-like extensions of the glands have a microtubular lining in the region where they penetrate the muscle layers, fibrous sheath and epithelium of the oesophagus. It is assumed that the tubules, together with the terminal septate desmosome,
525
strengthen and support the ducts in a region that is subjected to the muscular contractions of the feeding apparatus, and so allow a free flow of secretion to the gut lumen. The mechanism by which the secretion is discharged is not known, but the absence of intact secretory droplets in the oseophagus lumen suggests it is a merocrine type, involving membrane fusion. The actual release may be triggered by distension of the oesophagus lumen, such as would occur with the influx of food, so ensuring synchrony in the mixing of enzyme and substrate. Gland cells appear to be a distinguishing feature of the foregut of monogeneans. They have been recorded in light microscopic descriptions of other monopisthocotyleans, such as Entobdella solear and Acanthocotyle sp. by Kearn (1963) and E. hippoglossi by Halton & Jennings (1965) as well as for polyopisthocotyleans like Polystoma integerrimum by Williams (I 96l), Polystomoides sp. by Rohde (1974) and the gill flukes, Diclidophora merlangi, D. denticulata, Diplozoom paradoxum, Discocotyle sagittata and Plectanocotyle gurnardi by Halton & Jennings (1965) and Halton (1974). On the other hand, the anterior part of the alimentary tract of digenetic trematodes is generally nonglandular, with an extension of the body tegument lining not only the oral sucker but also the pharynx and oesophagus. In these worms, in contrast to the monogeneans so far examined by electron microscopy (Halton, 1975; Halton & Stranock, 1976), the cells of the caecal epithelium are both secretory and absorptive in function and provide the necessary hydrolytic enzymes for the extracellular phase of digestion (Halton, 1967; Howell, 1973 ; Robinson & Threadgold, 1975). The so-called oesophageal gland of Schistosoma, which appears to function in the intraluminar digestion of ingested blood cells, is in fact a modified form of oesophageal tegument, rather than a separate collection of gland cells (Morris & Threadgold. 1968; Spence & Silk, 1970; Dike, I97 I ). Achtro~ledgemrnl-apart of this work was undertaken while S.D.S. was in receipt of a Science Research Council Postgraduate Studentship.
REFERENCES S. C. 1971. Ultrastructure region in Schistosoma mansoni.
DIKE
Tropical
Medicine
and Hygiene
of the oesophageal American Journal 20: 552-568.
of
D. W. 1967. Observations on the nutrition of digenetic trematodes. Parasitology 57: 639-660. HALTON D. W. 1974. Functional aspects of monogenean gut cells. Proceedings of the 3rd Internariorlal Congrexv ofParasitology, Munich, August 1974, pp. 415-416. HALTON D. W. 1975. Intracellular digestion and cellular defecation in a monogenean, Diclidophora merlangi. HALTON
Parasitology
70: 331-340.
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D. W. HALTON and S. D. STRANO~K
HALTON D. W. & JENNINGS J. B. 1965. Observations on the nutrition of monogenetic trematodes. Bio/ogicuI Bulletin, Woods Hole, Massachusetts 129: 257-272. HALTON D. W., MORRIS G. P. & HARDCASTLE A. 1974. Gland cells associated with the alimentary tract of a monogenean, Diclidophora merlungi. Internationul Journal for Parasitology 4: 489-599. HALTON D. W. & MORRIS G. P. 1975. Ultrastructure of the anterior alimentary tract of a monogenean, Diclidophora merlungi. Internationul Journal for Parasifology 5 : 4074 19. HALTON D. W. & STRANOCK S. D. 1976. The line structure and histochemistry of the caecal epithelium of Calicoryle ltr6yeri (Monogenea: Monopisthocotylea). International Journal for Partritology 6: 253-263. HOWELL M. J. 1973. Localization of protcolytic activity in Fasciola hepatiea. The Journul c1.f’Pararitology 59: 454-456. KEARN C. C. 1963. Feeding in some monogencan skin parasites: Entobdella soleae on Solecr solea and Acanthocotyle sp. on Raia clavata. Journal of the Marine
1.J.P. VOL. 6. 1976
Biological Association of the United Kingdom 43: 749-766. MORRIS G. P. & THKEADGOLD L. T. 1968. Ultrastructure of the tegument of the adult Schistosoma mansoni. The Journal of Parasitology 54 : I 5-27. ROBINSON G. & THREADGOLD L. T. 1975. Electron microscope studies of Fasciola hepatica. XII. The fine structure of the gastrodermis. Experimental Parasitology 37: 20-36. ROI~DE K. 1974. Light and electron-microscopic studies of the pharynx and the anterior and posterior glands of Polystomoides (Monogenea: Folystomatidae). Zoologische Jahrbiicher Ahtreilung fiir Anatomie 92: l-17. SPENCE 1. M. & SILK M. H. 1970. Ultrastructural studies of the blood fluke~Schistosoma mansoni. IV. The digestive system. South African Journal of Medical Sciences 35: 93-112. WILLIAMS J. B. 1961. The dimorphism of Polystoma integerrimum (Friilich) Rudolphi and its bearing on relationships within the Polystomatidae: Part I. Journal of Helminthology 34: 151-192.