Inkmarional Journalfor Parasitology Vol. 22, No. 4, pp. 479-489, Printed in Great Britain
002&7Sl9/92 15.M) + 0.00 Pergomon Press Lid Auswalim Sociery for Pwosirology
1992
STUDIES ON THE ULTRASTRUCTURE AND HISTOCHEMISTRY THE LYMPH SYSTEM OF GASTRODISCOIDES HOMINIS (PARAMPHISTOMA: DIGENEA)
OF
G. P. BRENNAN,*~ R. E. B. HANNA* and W. A. NIZAMI~ *Division of Cell and Experimental Biology, School of Biology and Biochemistry, The Queen’s University of Belfast, Belfast BT9 5AG, Northern Ireland, U.K. $Section of Parasitology, Department of zoology, Aligarh Muslim University, Aligarh 202001, U.P., India (Received 28 August 1991; accepted 15 January 1992)
G. P., HANNAR. E. B. and NIZAMIW. A. 1992. Studies on the ultrastructure and histochemistry of the lymph system of Gustrodiscoides hominis (Paramphistoma: Digenea). International Journalfor Parasitology 22: 479489. The lymphatic system of the paramphistome, Gastrodiscoides hominis consists of numerous fluid-filled branches embedded in parenchyma and surrounded by extracellular material and is closely associated with the major organ systems of the fluke. The lymph matrix consists of a cytoplasmic syncytium within which nuclei, mitochondria and various sized granules and membranous structures occur. The granules found throughout the lymph system morphologically resemble autophagosomes and lysosomes. The lymph system provides a storage site for proteins which can be broken down to amino acids via autophagy, for subsequent mobilization and transport to tissues undergoing active protein synthesis. Many branches of the lymph system are surrounded by specialized parenchymal cells referred to as juxta-lymphatic cells. These cells are apparently associated with autophagic degradation of sequestered lymph cytoplasm, which may serve as an additional mechanism for the mobilization and transport of precursor molecules throughout the fluke via the parenchymal network. AbShICt-BRENNAN
INDEX KEY WORDS: Gustrodiscoides hominis; paramphistome; lymph ultrastructure; histochemistry; autophagy; amino acids; juxta-lymphatic cell.
INTRODUCTION PARAMPHISTOMES are widespread and abundant digenean parasites of domestic stock worldwide, especially in the tropics and sub-tropics, and are distinguished from other flukes by the possession of a posteriorly located acetabulum. Some families of trematodes are characterized by the possession of a series of interconnected, fluid-filled channels and sinuses, as described by Ozaki (1952) and Lowe (1966) for the Paramphistomidae, Balanorchidae, Cyclocoelidae and Angiodictyidae, and by Lowe (1966) for the monogenetic family Sphyranuridae. This is in addition to and independent of the excretory and gut systems. In live specimens, the contents of these channels are plasma-like and appear to ‘flow’ under the influence of specific peristaltic contractions of the lymph walls (Looss, 1902; Lowe, 1966; Rohde, 1962, 1963; Sharma & Ratnu, 1982) or due to general body movements (Stunkard, 1929; Willey, 1930, 1935; Ozaki, 1937). Looss (1902) proposed the term
t To whom all correspondence should be addressed.
system; mitochondria;
‘lymphgefassystem’ for these lymph channels because of their close association with the gut caeca and their similarity to lymph vessels in vertebrates. The contents of the lymph system have been described as lymphocytes by Willey (1930) in Pararnphistomum
stunkardi,
Diplodiscus
temperatus
and Cotylophoron cotylophorum and by Sharma & Ratnu (1982) in Orthocelium scolioelium, as haemocytoblasts by Jordon & Reynolds (1933) in D. temperatus, as haemocytes by Cheng & Streisfeld (1963) in Megalodiscus temperatus, or simply as cellular elements in Amphistomum spinzdosum and in five genera of Angiodictyidae by Looss (1902); and in C. cotylophorum by Stunkard (1929). Free-floating cells within the lymphatic matrix have been referred to as primitive phagocytes (Jordan & Reynolds, 1933; Cheng & Streisfeld, 1963; Sharma & Ratnu, 1982). However, Ozaki (1937, 1952) and Lowe (1966) reported the presence only of naked nuclei in the lymph of various Paramphistomum spp., with no evidence of free-floating cells. Various ‘granules’ and ‘particles’ have been frequently observed in the lymph matrix. Looss (1896, 1902) suggested that these 479
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G. P. BRENNAN, R. E. B. HANNAand W. A. NIZAMI
FIGS.1-4.
481
Lymph system of G. hominis fragments were food granules. Jordan & Reynolds (1933) considered them to be the products of phagocyte disintegration, while Dunn, Nizami & Hanna (1985) suggested that they resembled autophagosomes and lysosomes. In addition Dunn et al. (1985)‘identified endoplasmic reticulum and naked nuclei within the lymph matrix, however no distinct Golgi bodies or mitochondria were evident. Early electron microscopical observations of the lymph system are somewhat confusing. Strong & Bogitsh (1973) described it as possessing a lamellar epithelium, a feature reminiscent of the excretory system. The fine features of the lymph system, as observed in three species of amphistomes, were defined by Dunn et al. (1985) who suggested that it functions as a site for the storage and mobilization of amino acids and other small molecules. The aim of the present investigation was to examine the adult lymph system of the paramphistome, Gastrodiscoides hominis, using electron microscopical and histological techniques, in an attempt to describe its morphology and determine its function. MATERIALS AND METHODS Adult G. hominis collected from the pig abattoir in Aligarh, Uttar Pradesh, India, were processed for light microscopy and transmission electron microscopy (LM and TEM). For
general histochemistry, flukes were fixed in 10% neutral phosphate-buffered formalin, subsequently washed in buffer, dehydrated in ethanol and embedded in Paraplast wax (Agar Aids, Cambridge, U.K.). Sections (5 pm in thickness) were subjected to various histochemical tests as detailed in Table 1. For examination of general morphology, sections were stained with either haematoxylin and eosin or with periodic acid-Schiff (PAS) and orange G. For TEM, parasites were sliced transversely into three portions and fixed for 6 h at room temperature in 4% glutaraldehyde buffered with 0.1 Msodium cacodylate at pH 7.4. Subsequently, specimens were buffer-washed, subdivided into smaller pieces (approximately 1 mm’), post-fixed in 1% osmium tetroxide, dehydrated in
ethanol and embedded in TAAB resin (TAAB Labs, Reading, U.K.). Thin sections (60-70 nm) were cut with a diamond knife using a Reichert Ultracut E ultramicrotome, mounted on bare copper grids and double-stained with uranyl acetate and lead citrate. Sections were examined using a JEOL 1OOCX transmission electron microscope. RESULTS
Light microscope observations The appearance of Gastrodiscoides hominis under the light microscope clearly shows the gross arrangement of the major organ systems (Figs. l-3). The fluke is invested with a thick non-spinous tegumental syncytium which extends into the oral cone, with an underlying sub-tegumental musculature. Posterior to the oral sucker are the lateral pouches, the oesophagus and the oesophageal bulb, from which the paired lateral gut caeca extend posteriorly to the level of the acetabulum. No recognized pharynx was observed and, in the oesophageal bulb, valves were absent. The fluid-filled lymph system appears in very close association with the major organ systems of the fluke. The branches of this lymph system, which are embedded in the parenchyma and surrounded by an extracellular matrix, were frequently indistinguishable from the surrounding parenchymal cells in sections stained with toluidine blue or haematoxylin and eosin. However, this problem was largely overcome by staining the sectioned material with PAS and orange G. The lymph matrix incorporated the orange G and stained deep yellow while the parenchyma gave an intense PAS reaction (Fig. 2). The lymphatic system consists of a cytoplasmic syncytium in which naked nuclei and other various sized granules are frequently observed (Fig. 4). The nuclei occur singly or in groups, and the granules densely fill some branches of this lymph system while in others they are practically absent. Generally, the
FIG. 1. Light photomicrograph. Sag&al section stained with toluidine blue. Branches of the lymph system are present throughout the body and juxtapose organs such as the lateral pouches (Lp), the oesophageal bulb (Ob) and the testes (Te). A, Acetabulum; Ga, genital atrium. Scale bar, 266 pm. FIG. 2. Light photomicrograph. Longitudinal section stained with PAS and orange G. The parenchyma is PAS positive. Branches of the lymphatic system closely appose the gut caecum and are also associated with the reproductive organs, the acetabulum (A) and the tegument (T). Te, Testes; 0, ovary; Ob, oesophageal bulb; U, uterus; Lp, lateral pouch; L, lymph branch. Scale bar, 338 pm. FIG. 3. Light photomicrograph. Longitudinal section stained with haematoxylin and eosin highlighting some of the major organs of the fluke. MO, Oral sucker; 0, oesophagus; Ob, oesophageal bulb; Lp, lateral pouch; G, gut; L, lymph branch; T, tegument. Scale bar, 220 pm. FIG. 4. Light photomicrograph. Section showing minor branches of the lymphatic system (L) lying below the sub-tegumental musculature (St). The lymph matrix contains several nuclei (N). Scale bar, 34 pm.
482
G. P. BRENNAN,R. E. B. HANNAand W. A. NIZAMI TABLEI-HISTOCHEMICAL TESTS ONTHELYMPHATIC FLUID OFGartrodiscoides hominis
Method
Test for
Control
LD
Wax sections: Periodic acid/Schiff s
General carbohydrates
Amylase treated at 37°C for 1 h Amylase treated at 37’C for 1 h
_
Periodic acid/SchifP s with amylase at 37°C Periodic acid/Schiff s Orange G Mercuric bromophenol blue Methyl green Pyronin Y Pickworth’s benzidine method
Amylase-resistant carbohydrates Proteins and carbohydrates General proteins DNA RNA Haemogloblin
_ +++
Boiling water for 30 min
+ ++
1. Blood smear 2. Incubated at 130°C for 30 min
+++
Note: LD, lymph duct; + + + , intense activity; + + , moderate activity; + , mild activity; -, no activity. smaller branches of the lymph system occur in association with the major organ systems of the fluke, whereas the larger branches were mostly found in the deeper parenchyma. Histochemical studies (Table 1) show that the lymph matrix comprises high amounts of proteins and neutral lipids while carbohydrates are generally absent. The presence of haemoglobin, as denoted by the benzidine method was observed in the lymph system. Electron microscope observations At the ultrastructural level, the lymph appears as a dense, finely granular matrix within which occurs
occasional nuclei, mitochondria and various other membranous structures, together with numerous inclusion bodies (Fig. 5). The branches of the lymph system vary in shape and size, ranging from large dilated spaces (340 pm or more in diameter), to small branches (6 pm or less in diameter). In shape, the branches vary from those with smooth outlines to those with irregularly shaped outlines, with both types occasionally displaying narrow pseudopod-like extensions. The lymph system is bound by a unit membrane and separated from neighbouring cells by a layer of extracellular matrix. Embedded within this extracellular matrix are small bundles of muscle fibres. In many instances, the branches of the lymph system
FIG. 5. Transmission electron photomicrograph. The lymph matrix is a cytoplasmic syncytium containing nuclei and various other membranous structures (arrowed) some resembling ER-like material. L, Lymph matrix; N, nucleus; Nu, nucleolus. Scale bar, 1 pm. FIG. 6. Transmission electron photomicrograph. The lymph system is delimited by a bounding plasma membrane and is separated from neighbouring cells by a layer of extracellular matrix. I, Extracellular matrix; L. lymph matrix; Pm, plasma membrane. Scale bar, 2 pm. FIG. 7. Transmission electron photomicrograph. A juxta-lymphatic cell (JLC) lying adjacent to the lymph system. Spherical protrusions of lymph (L’) lie within the cytoplasm of the JLC which also contains a nucleus, and mitochondria. L, Lymph matrix; M, mitochondria; N, nucleus; Mu, muscle. Scale bar, 2 pm. FIG. 7a. Transmission electron photomicrograph. High magnification of the lymph protrusion into the JLC, as viewed in Fig. 7. JLC, Juxta-lymphatic cell; L’, lymph protrusion; M, mitochondria. Scale bar, 0.25 pm. FIG. 8. Transmission electron photomicrograph. The cytoplasmic lymph matrix exhibits primary (1,) and secondary (12) lysosome-like bodies. While 1, bodies are small and uniformly electron-dense, the 1, bodies have contents resembling the surrounding lymph cytoplasm or showing varying degrees of granularity. Primary lysosome-like bodies (1,) frequently associate with 1, bodies, possibly with membrane fusion (arrowed). L, Lymph matrix. Scale bar, 0.5 pm. FIG. 9. Transmission electron photomicrograph. Secondary lysosome-like bodies (12)are large and heteromorphic, sometimes containing electron-dense bodies resembling 1, bodies (arrowed). L, Lymph matrix. Scale bar, 0.2 pm.
Lymph system of G. hobby
FIGS. 5-9.
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G. P. BRENNAN,R. E. B. HANNAand W. A. NIZAMI
are surrounded by specialized parenchymal tissues referred to as juxta-lymphatic cells (Figs. 6, 7, 7a). These cells are characterized by numerous spherical protrusions of the lymph matrix into the peripheral cytoplasm which contains mitochondria, Golgi and nuclei with associated cisternae of endoplasmic reticulum (ER) and various inclusion bodies. Contents of lymphatic system The lymph system consists of a cytoplasmic syncytium in which naked nuclei occur frequently. Generally the nuclei are regularly shaped, ranging from 6 to 8 pm in diameter (Fig. 5). They appear singly or in groups of up to three or four nuclei. Cellular boundaries are absent between these nuclei and indeed elsewhere in the lymph system. Each nucleus contains a prominent nucleolus and dense heterochromatin. In many instances the nuclei are associated with an assortment of membranous structures some of which resemble ER-like material. A variety of other membrane-bound bodies is present within the lymph matrix. On the basis of their distribution, interrelationships and morphology, they are termed primary, secondary or tertiary lysosome-like bodies. Primary lysosome-like bodies possess a single limiting membrane and are small, spherical or elongate bodies with an electron-dense matrix. Primary lysosome-like bodies are randomly dispersed throughout the lymph system but, more frequently, are concentrated in areas where other inclusion bodies are absent (Fig. 8). Secondary lysosome-like bodies display a considerable range in size, shape and content. Most are larger than the primary lysosome-like bodies, heteromorphic and have a spherical or ovoid outline. The secondary lysosome-like bodies are generally delimited by a
double membrane but on occasions a single membrane or a number of concentric membranes were visible (Figs. 9-11). The internal matrix of most of the secondary lysosome-like bodies resembled the surrounding lymph matrix but was sometimes coarsely granular. Primary lysosome-like bodies were frequently observed in the vicinity of secondary lysosome-like bodies and occasionally fusion of the membranes of these structures was noted. Primary lysosome-like bodies were often observed in the matrix of the secondary lysosome-like bodies. Tertiary lysosome-like bodies (residual bodies) are oval, mostly regular structures with double or single limiting membranes (Fig. 12). These bodies vary in size but are generally smaller than the secondary lysosomelike bodies. These bodies, presumably loaded with residues of lysosomal digestion, are relatively dense and heterogeneous, and are characterized by the presence of myelin figures, electron-dense inclusions and droplets of an apparent lipid nature. Mitochondria were found in the lymph system. In many instances they occur singly or in pairs and occasionally they were found in groups (Fig. 13). They are delimited by a double membrane and contain distinct cristae. The mitochondria vary in shape and size but are mostly spherical (0.24.3 pm diameter) or elongate in outline. The inner mitochondrial membrane appears thicker than the outer membrane and the intermembranous space is generally of an even width. Lipid droplets were sometimes evident in the lymph system, but Golgi bodies were not observed in this study. Intercellular associations The branches of the lymph system are generally surrounded by parenchyma and occasionally small
FIG. 10. Transmission electron photomicrograph. Secondary lysosome-like bodies (12). The large structure containing a number of mitochondria is limited by a triple-layered membrane (arrow). These mitochondria show no apparent ultrastructural signs of degradation, however, they may represent early, perhaps still inactive, stages of autolysosomes or autophagosomes.
L, Lymph
matrix;
M, mitochondria.
Scale bar, 0.5 pm.
FIG. 11. Transmission electron photomicrograph. Secondary lysosome-like bodies (12). The cup-shaped membrane structure (arrowed), composed of two membranes, may be in the process of enclosing a portion of the lymph matrix, hence representing the earliest stage in the formation of an autolysosome or autophagosome. L, Lymph matrix. Scale bar, 0.5 pm. FIG. 12. Transmission electron photomicrograph. layered, myelin-like material. L, Lymph matrix; FIG. 13. Transmission
Tertiary lysosome-like bodies (13 are residual bodies containing abundant Me, myelin-like material; l,, secondary lysosome-like body. Scale bar, 2 pm.
electron photomicrograph. The lymph matrix exhibits numerous mitochondria. L, lymph matrix; M, mitochondria; Mu, muscle. Scale bar, 0.5 pm.
I, Extracellular
matrix;
FIG. 14. Transmission electron photomicrograph. Sequestered protrusions of lymph matrix (L’) lie within the cytoplasm of a JLC which also contains mitochondria and primary lysosome-like bodies (1,). The membranes of the lymph protrusions (arrowed) and the JLC are closely in contact, without intervening extracellular matrix. L, Lymph matrix; M. mitochondria. Scale bar, I pm.
Lymph system of G. hominis
FIGS. 10-14.
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G. P. BRENNAN.R. E. B. HANNAand W. A. NIZAMI
486
excretory tubules, muscle or nervous tissue juxtaposed them. However, the network of lymph branches which closely invests other major organs such as gut, reproductive organs and acetabulum is separated from these organ systems by an interposing layer of parenchyma. Intracellular associations were noted between the lymph system and the parenchyma. These comprise numerous spherical or pear-shaped protrusions of lymph into the peripheral cytoplasm of the adjacent juxta-lymphatic cells (Fig. 14). These membranebound protrusions are apparently ‘pinched off’ by laying down a membrane across the narrow junction, between the lymph and the juxta-lymphatic cell, thus enclosing them in the cytoplasm of the juxta-lymphatic cell. This junction continues to narrow until the sequestered lymph protrusion lies free in the cytoplasm of the cell. These membrane-bound protrusions of lymph were frequently observed surrounded by primary lysosome-like bodies. These possibly fuse with the limiting membrane of the protrusions. Large spherical or ovoid structures, possibly representing secondary lysosome-like bodies are also present within the juxta-lymphatic cells. They possess a finely granular matrix and contain varying amounts of dense material and electron¢ vacuoles. Other inclusions within the juxta-lymphatic cells resemble tertiary lysosomes. They are dense heterogeneous bodies varying in shape and size, some displaying lipid-like contents (Fig. 15). DISCUSSION
The present ultrastructural study has revealed that the lymph system of G. hominis conforms to earlier descriptions of the lymphatic system in various other amphistomes (Stunkard, 1929; Tandon, 1960a,b; Sharma & Ratnu, 1982; Dunn et al., 1985). A single pair of longitudinal ducts arise in the regions of the lateral pouches and extend posteriorly to the level of the acetabulum. These ducts give rise to numerous branches which form a close association with the major organ systems of the fluke, including the surface tegumental syncytium. However, no evidence was found for penetration of the lymph branches into the epithelium or tissues of any organ system. The lymphatic system did not openly communicate with the excretory system even though parts of both systems were often in very close contact. A layer of parenchyma was usually present between the branches of the lymph system and the juxtaposing organ. This finding is contrary to observations reported by other authors (Willey, 1930; Jordan & Reynolds, 1933; Lowe, 1966; Sharma & Ratnu, 1982). This ultrastructural study has also revealed that the branches of the lymph system are bound by a unit membrane and separated from neighbouring cells by a
layer of extracellular matrix. This also is contrary to the findings of most other investigators. Jordan & Reynolds (1933) and Tandon (1960a) suggested that the branches of the lymph system were lined with simple, flattened parenchyma cells. Lowe (1966) claimed that the walls of the lymph systems in two species of amphistome, Paramphistomum streptocoelium and Paramphistomum bathycotyle were composed of ‘connective fibres’ while in a third species, Paramphistomum calicophorum the walls were ‘entirely membranous’, an observation which agrees with the findings of Stunkard (1929), Willey (1930, 1935) Ozaki (1937, 1952) and Sharma & Ratnu (1982). Various authors have observed contractions of the walls of the lymph system independent to those of the body, suggesting the involvement of muscle fibres associated with branches of the lymph system, giving rise to pulsations of the lymph ducts accompanied by flowing movements of the lymph matrix (Looss, 1902; Rohde, 1962, 1963; Lowe, 1966; Sharma & Ratnu, 1982). In G. hominis, ultrastructural observations have indeed revealed the presence of small bundles of muscle fibres, embedded in an extracellular matrix, associated with the lymph system. Distinct cellular elements variously described as lymphocytes by Sharma & Ratnu (1982) haemocytoblasts by Jordan & Reynolds (1933) or haemocytes by Cheng & Streisfeld (1963) have been reported in the lymph system of a number of amphistome species and in other trematodes. The present study failed to reveal the presence of cellular elements in the lymph matrix. However, in agreement with the observations of Ozaki (1937, 1952), Lowe (1966) and Dunn et al. (1985) naked nuclei were encountered, occurring singly or in groups. In addition, mitochondria were also observed. This is the microscopic demonstration of first electron mitochondria in an amphistome lymph system. The mitochondria were found to occur singly or in groups, are elongate or ovoid in shape, varying in diameter, which ranged from 0.2 to 0.3 pm. Sharma (1978a,b) reported intense activity for the mitochondrial succinate dehydrogenase (SDH) and enzymes monoamine oxidase (MAO) within the lymph system of the amphistome CeyIonocotyle soliocoelium, with moderate activity in the tegument. However, he failed to demonstrate the presence of mitochondria. Clearly, confirmation of the presence of mitochondria requires further investigations by electron microscopical cytochemistry. The proteinaceous material present in the lymph matrix could be derived via polyribosomes since no organelles could clearly be identified as GER. Diffuse membranous areas of ER-like material associated
Lymph system of G. hominy
FIG. IS. Transmission electron photomicrograph. The cytoplasm of a JLC contains numerous 1, bodies which are heterogeneous, some with lipid-like contents. L, Lymph matrix; Lp, lipid-like material; JLC, juxta-lymphatic cell. Scale bar, 0.5 pm.
the lymph nuclei may possibly fulfil ER functions. Golgi bodies were also absent from the lymph matrix. Lysosomal production is generally associated with a Golgi complex, and although Golgi were not observed as such, areas of ER-like material may have Golgi activity. However, the demonstration of enzymes specific to the Golgi complex, such as thiamine pyrophosphatase, is necessary to determine the distribution and localization of these structures within the lymph matrix. In addition to these lymphatic inclusions, there were various other membranous structures, some of which appeared as sheets of membranes while others resembled residual bodies. The membrane sheets are apparently involved in sequestration of selected regions of the lymph matrix, enclosing it in a double membrane, possibly in preparation for autophagic degradation. Various other membrane-bound inclusions were present, possibly representing secondary lysosome-like bodies which, presumably, result from the addition of lysosomal hydrolases to autophagosomes. Following hydrolytic digestion, the autophagosomes become progressively transformed into residual bodies, containing indigestable material. Threadgold & Arme (1974) detailed the process of glycogenolysis in the parenchyma of F. hepatica. Evidence from ultrastructural observations suggests the existence of similar autophagic processes in the lymph system of G. hominis. However, the absence of observed Golgi suggests that not all the stages of autophagy, as described by these authors, were present with
in this study. Sequestration of selected regions of the lymph matrix, within autophagosomes, presumably gives rise to residual bodies suggesting the existence of an autophagic process which mobilizes amino acids for protein synthesis throughout the fluke. Threadgold & Anne (1974) suggested that parenchyma in F. hep~~~cuacts as a storage site for the mobili~tion of glycogen during times of carbohydrate shortage, while in G. hominis the lymph system acts as a storage site for proteins which can be mobilized and transported as amino acids and various other precursors throughout the body of the fluke. Many of the branches of the lymph system of G. hominis were shown to be surrounded by a specialized parenchymal cell type, the juxta-lymphatic cell (JLC). Ultrastructural evidence suggests that these cells are involved in autophagy of the contents of the lymph system. Numerous spherical protrusions of the lymph matrix into the peripheral cytoplasm of the adjoining JLCs were observed. These membrane-bound protrusions were then apparently ‘pinched off’ so as to lie free in the JLC cytoplasm, and then presumably fused with lysosome-like bodies, eventually giving rise to the formation of residual bodies, some with lipidlike contents. The reason for this autophagic breakdown of the lymph matrix by the JLCs is unclear. It may serve as an additional mechanism for the mobilization and transport of precursor molecules throughout the fluke via the parenchymal network. The presence of haemoglobin in the lymph system of G. hominis was localized cytochemically using the
488
G. P. BRENNAN. R. E. B. HANNA and W. A. NIZAMI
benzidine method. Haemoglobin has been recorded in the tissues of several other paramphistomes including Cotylophoron indicum by Goil (1961) and in Megulodiscus temperatus by Cain (1969). Haider & Siddiqi (1976) confirmed the occurrence of haemoglobin in G. hominis using spectrophotometry. The functional significance of haemoglobin in trematodes is not fully understood. Generally, haemoglobins have been implicated in oxygen storage and transport for respiratory processes (Lee & Smith, 1965). However, in some helminths they probably play a metabolic role other than in respiration (Smith & Lee, 1963). Previous studies have localized haemoglobin in areas of high metabolic activity, such as the oral and ventral suckers of Fasciofa gigantica (see Lutz 8~ Siddiqi, 1967) and the uterus of F. hepatica (Stephenson, 1947). In the present study the acetabulum and lateral pouches were found to be richly supplied by the lymph system. Hence, abundant haemoglobin is available near these regions of high metabolic activity. In general, parasite haemoglobins have a high affinity for oxygen, with saturation occurring at only 1 or 2 mm partial pressure (von Brand, 1952), which may confer advantages to parasites living in environments where anaerobic conditions prevail, or with low or fluctuating oxygen tensions (Lee & Smith, 1965), such as in the caecum and colon. For example, after ingestion of food and water the partial pressure of oxygen (PO,) within the caecum and colon may increase dramatically and the parasite haemoglobin binds oxygen, which can slowly be released in conditions of declining oxygen levels, to facilitate nerve function, etc. Confirmation of the role of haemoglobin in paramphistomes requires extensive physiological investigation, nevertheless, it is likely that they function in oxygen storage and transport. Clearly, the lymph system and its structural relationship with other major organ systems of the require further detailed investigations fluke incorporating histochemical, cytochemical and electron microscopical studies. REFERENCES BRAND T. VON 1952. Chemical Physiology of Endoparasitic Animals. Academic Press, New York. CAIN G. D. 1969. Studies on haemoglobins in some digenetic trematodes. Journal of Parasitology 55: 301-306. CHENGT. C. & S-~REISFELD S. D. 1963. Innate phagocytosis in the trematodes Megalodiscus temperatus and Haematoloechus sp. Journal of Morphology 113: 375-380. DUNN T. S.. NIZAMI W. A. & HANNA R. E. B. 1985. Studies on the ultrastructure and histochemistry of the lymph system of three species of amphistome (Trematoda: Digenea), Gigantocotyle explanatum, Gastrothylax crumenifer and
Srivastavaia indica from the Indian Water Buffalo Bubalus bubalis. Journal of Helminthology 59: 1-18. GAIL H. S. 1961. Haemoglobin in trematodes 1. Fasciola gigantica 2. Cotylophoron indiclim. Zeitschrift fiir Parasitenkunde 201 572-575. HAIDER A. S. & SIDDIQI A. H. 1976. Spectrophotometric analysis of haemoglobins of some digenetic trematodes and their hosts. Journal of Helminthology 50: 259-266. JORWN H. E. & REYNOLDSB. D. 1933. The blood cells of the trematode Diplodiscus temperatus. Journal of Morphology 55: 119-130. LEE D. L. & SMITH M. H. 1965. Haemoglobins of parasitic animals. Experimental Parasitology 16: 392-424. Looss A. 1896. Recherches sur la faune parasitaire de
I’Egypte. Premiire partie. Mtmoires de 1’Institut Egyptien. Le Caire 3: 1-252. Looss A. 1902. Uber neue und bekannte Trematoden aus SeeschildkrBten. Zoologische Jahrbiicher. Abteilung fiir Systematik, Okologie und Geographie der Tiere 16: 41 l-
418. LOWE C. Y. 1966. Comparative studies of the lymphatic system of four species of amphistomes. Zeitschrifr fiir Parasitenkunde 27: 169-204. LUTZ P. L. & SIDDIQI A. H. 1967. Comparison of haemoglobins of Fasciola gigantica (Trematoda: Digenea) and its host. Experimental Parasitology 20: 83-87. OZAKI Y. 1937. Studies on the trematode families Gyliauchenidae and Opistholebetidae, with special reference to lymph system. I and II. Journal of Science of Hiroshima University 5: 125-244. OZAKI Y. 1952. Lymph system of Paramphistomum orthocoelium and other two species. Journal of Science of Hiroshima University 13: 79-84. ROHDE K. 1962. Parorientodiscus magnus n.g., n.sp. ein Trematode aus dem Darm von Cyclemys amboinensis (Daud) in Malaya. Zeitschrift fcr Parasitenkunde 21: 457464. ROHDE K. 1963. Orientodiscus fernandoi n.sp. and 0. hendricksoni n.sp. (Trematoda, Paramphistomata) from the intestine of Trionyx spp. in Malaya. Journal of Helminthology 31: 349-358. SHARMAP. N. 1978a. Histochemical localization of succinate dehydrogenase in the lymphatic system of a trematode Ceylonocotyle scoliocoelium. Journal of Helminthology 52: 159-162. SHARMA P. N. 1978b. Histochemical observations on the distribution of monoamine oxidase in the lymphatic system of the Ceylonocotyle scoliocoelium amphistome Fishchoeder 1901 (Trematoda: Digenea). Indian Journalof Experimental Biology 16: 1202-1203. SHARMA P. N. & RATNU L. S. 1982. Morphology, histochemistry and biological significance of the lymphatic system of the trematode Orchocoelium scoliocoelium. Journal of Helminthology 56: 59-67. SMITH M. H. & LEE D. L. 1963. Metabolism of haemoglobin and haematin compounds in Ascaris lumbricoides. Proceedings of the Royal Society, Series B 157: 234-257. STEPHENSON W. 1947. Physiological and histochemical observations on the adult liver fluke Fasciola hepatica L. III. Egg shell formation. Parasitology 38: 128-139.
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system of G. hominis
STRONG P. A. & BOGITSH B. J. 1973. Ultrastructure of the lymph system of the trematode Megalodiscus temperatus. Transactions of the American Microscopical Society 92: 570-578. STUNKARD H. W. 1929. The parasitic worms collected by the American Museum of Natural History Expedition to the Belgian Congo, 190991914. Bulletin of the American Museum of Natural History 58: 233-289. TANDON R. S. 1960a. Studies on the lymphatic system of amphistomes of ruminants: I. Carmyerius spatiosus (Stiles & Goldberger. 1910). Zoologischer Anzeiger 159: 213-217. TANDON R. S. 1960b. Studies on the lymphatic system of
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amphistomes of ruminants: 2. The genera Gastrothylax and Fischoederius. Zoologischer Anzeiger 159:2 17-22 1. THREADGOLDL. T. & ARME C. 1974. Electron microscope studies of Fasciola hepatica XI. Autophagy and parenchymal cell function. Experimental Parasitology 35: 389-405. WILLEY C. H. 1930. Studies on the lymph system of digenetic trematodes. Journal of Morphology and Ph.vsiologySO: l-37. WILLEY C. H. 1935. The excretory system of the trematode, Typhiocoelum cucumerinum, with notes on lymph-like structures in the family Cyclocoelidae. Journal of Morphology 57: 461471.
002&7Sl9/92 15.M) + 0.00 Pergomon Press Lid Auswalim Sociery for Pwosirology
1992
STUDIES ON THE ULTRASTRUCTURE AND HISTOCHEMISTRY THE LYMPH SYSTEM OF GASTRODISCOIDES HOMINIS (PARAMPHISTOMA: DIGENEA)
OF
G. P. BRENNAN,*~ R. E. B. HANNA* and W. A. NIZAMI~ *Division of Cell and Experimental Biology, School of Biology and Biochemistry, The Queen’s University of Belfast, Belfast BT9 5AG, Northern Ireland, U.K. $Section of Parasitology, Department of zoology, Aligarh Muslim University, Aligarh 202001, U.P., India (Received 28 August 1991; accepted 15 January 1992)
G. P., HANNAR. E. B. and NIZAMIW. A. 1992. Studies on the ultrastructure and histochemistry of the lymph system of Gustrodiscoides hominis (Paramphistoma: Digenea). International Journalfor Parasitology 22: 479489. The lymphatic system of the paramphistome, Gastrodiscoides hominis consists of numerous fluid-filled branches embedded in parenchyma and surrounded by extracellular material and is closely associated with the major organ systems of the fluke. The lymph matrix consists of a cytoplasmic syncytium within which nuclei, mitochondria and various sized granules and membranous structures occur. The granules found throughout the lymph system morphologically resemble autophagosomes and lysosomes. The lymph system provides a storage site for proteins which can be broken down to amino acids via autophagy, for subsequent mobilization and transport to tissues undergoing active protein synthesis. Many branches of the lymph system are surrounded by specialized parenchymal cells referred to as juxta-lymphatic cells. These cells are apparently associated with autophagic degradation of sequestered lymph cytoplasm, which may serve as an additional mechanism for the mobilization and transport of precursor molecules throughout the fluke via the parenchymal network. AbShICt-BRENNAN
INDEX KEY WORDS: Gustrodiscoides hominis; paramphistome; lymph ultrastructure; histochemistry; autophagy; amino acids; juxta-lymphatic cell.
INTRODUCTION PARAMPHISTOMES are widespread and abundant digenean parasites of domestic stock worldwide, especially in the tropics and sub-tropics, and are distinguished from other flukes by the possession of a posteriorly located acetabulum. Some families of trematodes are characterized by the possession of a series of interconnected, fluid-filled channels and sinuses, as described by Ozaki (1952) and Lowe (1966) for the Paramphistomidae, Balanorchidae, Cyclocoelidae and Angiodictyidae, and by Lowe (1966) for the monogenetic family Sphyranuridae. This is in addition to and independent of the excretory and gut systems. In live specimens, the contents of these channels are plasma-like and appear to ‘flow’ under the influence of specific peristaltic contractions of the lymph walls (Looss, 1902; Lowe, 1966; Rohde, 1962, 1963; Sharma & Ratnu, 1982) or due to general body movements (Stunkard, 1929; Willey, 1930, 1935; Ozaki, 1937). Looss (1902) proposed the term
t To whom all correspondence should be addressed.
system; mitochondria;
‘lymphgefassystem’ for these lymph channels because of their close association with the gut caeca and their similarity to lymph vessels in vertebrates. The contents of the lymph system have been described as lymphocytes by Willey (1930) in Pararnphistomum
stunkardi,
Diplodiscus
temperatus
and Cotylophoron cotylophorum and by Sharma & Ratnu (1982) in Orthocelium scolioelium, as haemocytoblasts by Jordon & Reynolds (1933) in D. temperatus, as haemocytes by Cheng & Streisfeld (1963) in Megalodiscus temperatus, or simply as cellular elements in Amphistomum spinzdosum and in five genera of Angiodictyidae by Looss (1902); and in C. cotylophorum by Stunkard (1929). Free-floating cells within the lymphatic matrix have been referred to as primitive phagocytes (Jordan & Reynolds, 1933; Cheng & Streisfeld, 1963; Sharma & Ratnu, 1982). However, Ozaki (1937, 1952) and Lowe (1966) reported the presence only of naked nuclei in the lymph of various Paramphistomum spp., with no evidence of free-floating cells. Various ‘granules’ and ‘particles’ have been frequently observed in the lymph matrix. Looss (1896, 1902) suggested that these 479
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G. P. BRENNAN, R. E. B. HANNAand W. A. NIZAMI
FIGS.1-4.
481
Lymph system of G. hominis fragments were food granules. Jordan & Reynolds (1933) considered them to be the products of phagocyte disintegration, while Dunn, Nizami & Hanna (1985) suggested that they resembled autophagosomes and lysosomes. In addition Dunn et al. (1985)‘identified endoplasmic reticulum and naked nuclei within the lymph matrix, however no distinct Golgi bodies or mitochondria were evident. Early electron microscopical observations of the lymph system are somewhat confusing. Strong & Bogitsh (1973) described it as possessing a lamellar epithelium, a feature reminiscent of the excretory system. The fine features of the lymph system, as observed in three species of amphistomes, were defined by Dunn et al. (1985) who suggested that it functions as a site for the storage and mobilization of amino acids and other small molecules. The aim of the present investigation was to examine the adult lymph system of the paramphistome, Gastrodiscoides hominis, using electron microscopical and histological techniques, in an attempt to describe its morphology and determine its function. MATERIALS AND METHODS Adult G. hominis collected from the pig abattoir in Aligarh, Uttar Pradesh, India, were processed for light microscopy and transmission electron microscopy (LM and TEM). For
general histochemistry, flukes were fixed in 10% neutral phosphate-buffered formalin, subsequently washed in buffer, dehydrated in ethanol and embedded in Paraplast wax (Agar Aids, Cambridge, U.K.). Sections (5 pm in thickness) were subjected to various histochemical tests as detailed in Table 1. For examination of general morphology, sections were stained with either haematoxylin and eosin or with periodic acid-Schiff (PAS) and orange G. For TEM, parasites were sliced transversely into three portions and fixed for 6 h at room temperature in 4% glutaraldehyde buffered with 0.1 Msodium cacodylate at pH 7.4. Subsequently, specimens were buffer-washed, subdivided into smaller pieces (approximately 1 mm’), post-fixed in 1% osmium tetroxide, dehydrated in
ethanol and embedded in TAAB resin (TAAB Labs, Reading, U.K.). Thin sections (60-70 nm) were cut with a diamond knife using a Reichert Ultracut E ultramicrotome, mounted on bare copper grids and double-stained with uranyl acetate and lead citrate. Sections were examined using a JEOL 1OOCX transmission electron microscope. RESULTS
Light microscope observations The appearance of Gastrodiscoides hominis under the light microscope clearly shows the gross arrangement of the major organ systems (Figs. l-3). The fluke is invested with a thick non-spinous tegumental syncytium which extends into the oral cone, with an underlying sub-tegumental musculature. Posterior to the oral sucker are the lateral pouches, the oesophagus and the oesophageal bulb, from which the paired lateral gut caeca extend posteriorly to the level of the acetabulum. No recognized pharynx was observed and, in the oesophageal bulb, valves were absent. The fluid-filled lymph system appears in very close association with the major organ systems of the fluke. The branches of this lymph system, which are embedded in the parenchyma and surrounded by an extracellular matrix, were frequently indistinguishable from the surrounding parenchymal cells in sections stained with toluidine blue or haematoxylin and eosin. However, this problem was largely overcome by staining the sectioned material with PAS and orange G. The lymph matrix incorporated the orange G and stained deep yellow while the parenchyma gave an intense PAS reaction (Fig. 2). The lymphatic system consists of a cytoplasmic syncytium in which naked nuclei and other various sized granules are frequently observed (Fig. 4). The nuclei occur singly or in groups, and the granules densely fill some branches of this lymph system while in others they are practically absent. Generally, the
FIG. 1. Light photomicrograph. Sag&al section stained with toluidine blue. Branches of the lymph system are present throughout the body and juxtapose organs such as the lateral pouches (Lp), the oesophageal bulb (Ob) and the testes (Te). A, Acetabulum; Ga, genital atrium. Scale bar, 266 pm. FIG. 2. Light photomicrograph. Longitudinal section stained with PAS and orange G. The parenchyma is PAS positive. Branches of the lymphatic system closely appose the gut caecum and are also associated with the reproductive organs, the acetabulum (A) and the tegument (T). Te, Testes; 0, ovary; Ob, oesophageal bulb; U, uterus; Lp, lateral pouch; L, lymph branch. Scale bar, 338 pm. FIG. 3. Light photomicrograph. Longitudinal section stained with haematoxylin and eosin highlighting some of the major organs of the fluke. MO, Oral sucker; 0, oesophagus; Ob, oesophageal bulb; Lp, lateral pouch; G, gut; L, lymph branch; T, tegument. Scale bar, 220 pm. FIG. 4. Light photomicrograph. Section showing minor branches of the lymphatic system (L) lying below the sub-tegumental musculature (St). The lymph matrix contains several nuclei (N). Scale bar, 34 pm.
482
G. P. BRENNAN,R. E. B. HANNAand W. A. NIZAMI TABLEI-HISTOCHEMICAL TESTS ONTHELYMPHATIC FLUID OFGartrodiscoides hominis
Method
Test for
Control
LD
Wax sections: Periodic acid/Schiff s
General carbohydrates
Amylase treated at 37°C for 1 h Amylase treated at 37’C for 1 h
_
Periodic acid/SchifP s with amylase at 37°C Periodic acid/Schiff s Orange G Mercuric bromophenol blue Methyl green Pyronin Y Pickworth’s benzidine method
Amylase-resistant carbohydrates Proteins and carbohydrates General proteins DNA RNA Haemogloblin
_ +++
Boiling water for 30 min
+ ++
1. Blood smear 2. Incubated at 130°C for 30 min
+++
Note: LD, lymph duct; + + + , intense activity; + + , moderate activity; + , mild activity; -, no activity. smaller branches of the lymph system occur in association with the major organ systems of the fluke, whereas the larger branches were mostly found in the deeper parenchyma. Histochemical studies (Table 1) show that the lymph matrix comprises high amounts of proteins and neutral lipids while carbohydrates are generally absent. The presence of haemoglobin, as denoted by the benzidine method was observed in the lymph system. Electron microscope observations At the ultrastructural level, the lymph appears as a dense, finely granular matrix within which occurs
occasional nuclei, mitochondria and various other membranous structures, together with numerous inclusion bodies (Fig. 5). The branches of the lymph system vary in shape and size, ranging from large dilated spaces (340 pm or more in diameter), to small branches (6 pm or less in diameter). In shape, the branches vary from those with smooth outlines to those with irregularly shaped outlines, with both types occasionally displaying narrow pseudopod-like extensions. The lymph system is bound by a unit membrane and separated from neighbouring cells by a layer of extracellular matrix. Embedded within this extracellular matrix are small bundles of muscle fibres. In many instances, the branches of the lymph system
FIG. 5. Transmission electron photomicrograph. The lymph matrix is a cytoplasmic syncytium containing nuclei and various other membranous structures (arrowed) some resembling ER-like material. L, Lymph matrix; N, nucleus; Nu, nucleolus. Scale bar, 1 pm. FIG. 6. Transmission electron photomicrograph. The lymph system is delimited by a bounding plasma membrane and is separated from neighbouring cells by a layer of extracellular matrix. I, Extracellular matrix; L. lymph matrix; Pm, plasma membrane. Scale bar, 2 pm. FIG. 7. Transmission electron photomicrograph. A juxta-lymphatic cell (JLC) lying adjacent to the lymph system. Spherical protrusions of lymph (L’) lie within the cytoplasm of the JLC which also contains a nucleus, and mitochondria. L, Lymph matrix; M, mitochondria; N, nucleus; Mu, muscle. Scale bar, 2 pm. FIG. 7a. Transmission electron photomicrograph. High magnification of the lymph protrusion into the JLC, as viewed in Fig. 7. JLC, Juxta-lymphatic cell; L’, lymph protrusion; M, mitochondria. Scale bar, 0.25 pm. FIG. 8. Transmission electron photomicrograph. The cytoplasmic lymph matrix exhibits primary (1,) and secondary (12) lysosome-like bodies. While 1, bodies are small and uniformly electron-dense, the 1, bodies have contents resembling the surrounding lymph cytoplasm or showing varying degrees of granularity. Primary lysosome-like bodies (1,) frequently associate with 1, bodies, possibly with membrane fusion (arrowed). L, Lymph matrix. Scale bar, 0.5 pm. FIG. 9. Transmission electron photomicrograph. Secondary lysosome-like bodies (12)are large and heteromorphic, sometimes containing electron-dense bodies resembling 1, bodies (arrowed). L, Lymph matrix. Scale bar, 0.2 pm.
Lymph system of G. hobby
FIGS. 5-9.
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G. P. BRENNAN,R. E. B. HANNAand W. A. NIZAMI
are surrounded by specialized parenchymal tissues referred to as juxta-lymphatic cells (Figs. 6, 7, 7a). These cells are characterized by numerous spherical protrusions of the lymph matrix into the peripheral cytoplasm which contains mitochondria, Golgi and nuclei with associated cisternae of endoplasmic reticulum (ER) and various inclusion bodies. Contents of lymphatic system The lymph system consists of a cytoplasmic syncytium in which naked nuclei occur frequently. Generally the nuclei are regularly shaped, ranging from 6 to 8 pm in diameter (Fig. 5). They appear singly or in groups of up to three or four nuclei. Cellular boundaries are absent between these nuclei and indeed elsewhere in the lymph system. Each nucleus contains a prominent nucleolus and dense heterochromatin. In many instances the nuclei are associated with an assortment of membranous structures some of which resemble ER-like material. A variety of other membrane-bound bodies is present within the lymph matrix. On the basis of their distribution, interrelationships and morphology, they are termed primary, secondary or tertiary lysosome-like bodies. Primary lysosome-like bodies possess a single limiting membrane and are small, spherical or elongate bodies with an electron-dense matrix. Primary lysosome-like bodies are randomly dispersed throughout the lymph system but, more frequently, are concentrated in areas where other inclusion bodies are absent (Fig. 8). Secondary lysosome-like bodies display a considerable range in size, shape and content. Most are larger than the primary lysosome-like bodies, heteromorphic and have a spherical or ovoid outline. The secondary lysosome-like bodies are generally delimited by a
double membrane but on occasions a single membrane or a number of concentric membranes were visible (Figs. 9-11). The internal matrix of most of the secondary lysosome-like bodies resembled the surrounding lymph matrix but was sometimes coarsely granular. Primary lysosome-like bodies were frequently observed in the vicinity of secondary lysosome-like bodies and occasionally fusion of the membranes of these structures was noted. Primary lysosome-like bodies were often observed in the matrix of the secondary lysosome-like bodies. Tertiary lysosome-like bodies (residual bodies) are oval, mostly regular structures with double or single limiting membranes (Fig. 12). These bodies vary in size but are generally smaller than the secondary lysosomelike bodies. These bodies, presumably loaded with residues of lysosomal digestion, are relatively dense and heterogeneous, and are characterized by the presence of myelin figures, electron-dense inclusions and droplets of an apparent lipid nature. Mitochondria were found in the lymph system. In many instances they occur singly or in pairs and occasionally they were found in groups (Fig. 13). They are delimited by a double membrane and contain distinct cristae. The mitochondria vary in shape and size but are mostly spherical (0.24.3 pm diameter) or elongate in outline. The inner mitochondrial membrane appears thicker than the outer membrane and the intermembranous space is generally of an even width. Lipid droplets were sometimes evident in the lymph system, but Golgi bodies were not observed in this study. Intercellular associations The branches of the lymph system are generally surrounded by parenchyma and occasionally small
FIG. 10. Transmission electron photomicrograph. Secondary lysosome-like bodies (12). The large structure containing a number of mitochondria is limited by a triple-layered membrane (arrow). These mitochondria show no apparent ultrastructural signs of degradation, however, they may represent early, perhaps still inactive, stages of autolysosomes or autophagosomes.
L, Lymph
matrix;
M, mitochondria.
Scale bar, 0.5 pm.
FIG. 11. Transmission electron photomicrograph. Secondary lysosome-like bodies (12). The cup-shaped membrane structure (arrowed), composed of two membranes, may be in the process of enclosing a portion of the lymph matrix, hence representing the earliest stage in the formation of an autolysosome or autophagosome. L, Lymph matrix. Scale bar, 0.5 pm. FIG. 12. Transmission electron photomicrograph. layered, myelin-like material. L, Lymph matrix; FIG. 13. Transmission
Tertiary lysosome-like bodies (13 are residual bodies containing abundant Me, myelin-like material; l,, secondary lysosome-like body. Scale bar, 2 pm.
electron photomicrograph. The lymph matrix exhibits numerous mitochondria. L, lymph matrix; M, mitochondria; Mu, muscle. Scale bar, 0.5 pm.
I, Extracellular
matrix;
FIG. 14. Transmission electron photomicrograph. Sequestered protrusions of lymph matrix (L’) lie within the cytoplasm of a JLC which also contains mitochondria and primary lysosome-like bodies (1,). The membranes of the lymph protrusions (arrowed) and the JLC are closely in contact, without intervening extracellular matrix. L, Lymph matrix; M. mitochondria. Scale bar, I pm.
Lymph system of G. hominis
FIGS. 10-14.
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G. P. BRENNAN.R. E. B. HANNAand W. A. NIZAMI
486
excretory tubules, muscle or nervous tissue juxtaposed them. However, the network of lymph branches which closely invests other major organs such as gut, reproductive organs and acetabulum is separated from these organ systems by an interposing layer of parenchyma. Intracellular associations were noted between the lymph system and the parenchyma. These comprise numerous spherical or pear-shaped protrusions of lymph into the peripheral cytoplasm of the adjacent juxta-lymphatic cells (Fig. 14). These membranebound protrusions are apparently ‘pinched off’ by laying down a membrane across the narrow junction, between the lymph and the juxta-lymphatic cell, thus enclosing them in the cytoplasm of the juxta-lymphatic cell. This junction continues to narrow until the sequestered lymph protrusion lies free in the cytoplasm of the cell. These membrane-bound protrusions of lymph were frequently observed surrounded by primary lysosome-like bodies. These possibly fuse with the limiting membrane of the protrusions. Large spherical or ovoid structures, possibly representing secondary lysosome-like bodies are also present within the juxta-lymphatic cells. They possess a finely granular matrix and contain varying amounts of dense material and electron¢ vacuoles. Other inclusions within the juxta-lymphatic cells resemble tertiary lysosomes. They are dense heterogeneous bodies varying in shape and size, some displaying lipid-like contents (Fig. 15). DISCUSSION
The present ultrastructural study has revealed that the lymph system of G. hominis conforms to earlier descriptions of the lymphatic system in various other amphistomes (Stunkard, 1929; Tandon, 1960a,b; Sharma & Ratnu, 1982; Dunn et al., 1985). A single pair of longitudinal ducts arise in the regions of the lateral pouches and extend posteriorly to the level of the acetabulum. These ducts give rise to numerous branches which form a close association with the major organ systems of the fluke, including the surface tegumental syncytium. However, no evidence was found for penetration of the lymph branches into the epithelium or tissues of any organ system. The lymphatic system did not openly communicate with the excretory system even though parts of both systems were often in very close contact. A layer of parenchyma was usually present between the branches of the lymph system and the juxtaposing organ. This finding is contrary to observations reported by other authors (Willey, 1930; Jordan & Reynolds, 1933; Lowe, 1966; Sharma & Ratnu, 1982). This ultrastructural study has also revealed that the branches of the lymph system are bound by a unit membrane and separated from neighbouring cells by a
layer of extracellular matrix. This also is contrary to the findings of most other investigators. Jordan & Reynolds (1933) and Tandon (1960a) suggested that the branches of the lymph system were lined with simple, flattened parenchyma cells. Lowe (1966) claimed that the walls of the lymph systems in two species of amphistome, Paramphistomum streptocoelium and Paramphistomum bathycotyle were composed of ‘connective fibres’ while in a third species, Paramphistomum calicophorum the walls were ‘entirely membranous’, an observation which agrees with the findings of Stunkard (1929), Willey (1930, 1935) Ozaki (1937, 1952) and Sharma & Ratnu (1982). Various authors have observed contractions of the walls of the lymph system independent to those of the body, suggesting the involvement of muscle fibres associated with branches of the lymph system, giving rise to pulsations of the lymph ducts accompanied by flowing movements of the lymph matrix (Looss, 1902; Rohde, 1962, 1963; Lowe, 1966; Sharma & Ratnu, 1982). In G. hominis, ultrastructural observations have indeed revealed the presence of small bundles of muscle fibres, embedded in an extracellular matrix, associated with the lymph system. Distinct cellular elements variously described as lymphocytes by Sharma & Ratnu (1982) haemocytoblasts by Jordan & Reynolds (1933) or haemocytes by Cheng & Streisfeld (1963) have been reported in the lymph system of a number of amphistome species and in other trematodes. The present study failed to reveal the presence of cellular elements in the lymph matrix. However, in agreement with the observations of Ozaki (1937, 1952), Lowe (1966) and Dunn et al. (1985) naked nuclei were encountered, occurring singly or in groups. In addition, mitochondria were also observed. This is the microscopic demonstration of first electron mitochondria in an amphistome lymph system. The mitochondria were found to occur singly or in groups, are elongate or ovoid in shape, varying in diameter, which ranged from 0.2 to 0.3 pm. Sharma (1978a,b) reported intense activity for the mitochondrial succinate dehydrogenase (SDH) and enzymes monoamine oxidase (MAO) within the lymph system of the amphistome CeyIonocotyle soliocoelium, with moderate activity in the tegument. However, he failed to demonstrate the presence of mitochondria. Clearly, confirmation of the presence of mitochondria requires further investigations by electron microscopical cytochemistry. The proteinaceous material present in the lymph matrix could be derived via polyribosomes since no organelles could clearly be identified as GER. Diffuse membranous areas of ER-like material associated
Lymph system of G. hominy
FIG. IS. Transmission electron photomicrograph. The cytoplasm of a JLC contains numerous 1, bodies which are heterogeneous, some with lipid-like contents. L, Lymph matrix; Lp, lipid-like material; JLC, juxta-lymphatic cell. Scale bar, 0.5 pm.
the lymph nuclei may possibly fulfil ER functions. Golgi bodies were also absent from the lymph matrix. Lysosomal production is generally associated with a Golgi complex, and although Golgi were not observed as such, areas of ER-like material may have Golgi activity. However, the demonstration of enzymes specific to the Golgi complex, such as thiamine pyrophosphatase, is necessary to determine the distribution and localization of these structures within the lymph matrix. In addition to these lymphatic inclusions, there were various other membranous structures, some of which appeared as sheets of membranes while others resembled residual bodies. The membrane sheets are apparently involved in sequestration of selected regions of the lymph matrix, enclosing it in a double membrane, possibly in preparation for autophagic degradation. Various other membrane-bound inclusions were present, possibly representing secondary lysosome-like bodies which, presumably, result from the addition of lysosomal hydrolases to autophagosomes. Following hydrolytic digestion, the autophagosomes become progressively transformed into residual bodies, containing indigestable material. Threadgold & Arme (1974) detailed the process of glycogenolysis in the parenchyma of F. hepatica. Evidence from ultrastructural observations suggests the existence of similar autophagic processes in the lymph system of G. hominis. However, the absence of observed Golgi suggests that not all the stages of autophagy, as described by these authors, were present with
in this study. Sequestration of selected regions of the lymph matrix, within autophagosomes, presumably gives rise to residual bodies suggesting the existence of an autophagic process which mobilizes amino acids for protein synthesis throughout the fluke. Threadgold & Anne (1974) suggested that parenchyma in F. hep~~~cuacts as a storage site for the mobili~tion of glycogen during times of carbohydrate shortage, while in G. hominis the lymph system acts as a storage site for proteins which can be mobilized and transported as amino acids and various other precursors throughout the body of the fluke. Many of the branches of the lymph system of G. hominis were shown to be surrounded by a specialized parenchymal cell type, the juxta-lymphatic cell (JLC). Ultrastructural evidence suggests that these cells are involved in autophagy of the contents of the lymph system. Numerous spherical protrusions of the lymph matrix into the peripheral cytoplasm of the adjoining JLCs were observed. These membrane-bound protrusions were then apparently ‘pinched off’ so as to lie free in the JLC cytoplasm, and then presumably fused with lysosome-like bodies, eventually giving rise to the formation of residual bodies, some with lipidlike contents. The reason for this autophagic breakdown of the lymph matrix by the JLCs is unclear. It may serve as an additional mechanism for the mobilization and transport of precursor molecules throughout the fluke via the parenchymal network. The presence of haemoglobin in the lymph system of G. hominis was localized cytochemically using the
488
G. P. BRENNAN. R. E. B. HANNA and W. A. NIZAMI
benzidine method. Haemoglobin has been recorded in the tissues of several other paramphistomes including Cotylophoron indicum by Goil (1961) and in Megulodiscus temperatus by Cain (1969). Haider & Siddiqi (1976) confirmed the occurrence of haemoglobin in G. hominis using spectrophotometry. The functional significance of haemoglobin in trematodes is not fully understood. Generally, haemoglobins have been implicated in oxygen storage and transport for respiratory processes (Lee & Smith, 1965). However, in some helminths they probably play a metabolic role other than in respiration (Smith & Lee, 1963). Previous studies have localized haemoglobin in areas of high metabolic activity, such as the oral and ventral suckers of Fasciofa gigantica (see Lutz 8~ Siddiqi, 1967) and the uterus of F. hepatica (Stephenson, 1947). In the present study the acetabulum and lateral pouches were found to be richly supplied by the lymph system. Hence, abundant haemoglobin is available near these regions of high metabolic activity. In general, parasite haemoglobins have a high affinity for oxygen, with saturation occurring at only 1 or 2 mm partial pressure (von Brand, 1952), which may confer advantages to parasites living in environments where anaerobic conditions prevail, or with low or fluctuating oxygen tensions (Lee & Smith, 1965), such as in the caecum and colon. For example, after ingestion of food and water the partial pressure of oxygen (PO,) within the caecum and colon may increase dramatically and the parasite haemoglobin binds oxygen, which can slowly be released in conditions of declining oxygen levels, to facilitate nerve function, etc. Confirmation of the role of haemoglobin in paramphistomes requires extensive physiological investigation, nevertheless, it is likely that they function in oxygen storage and transport. Clearly, the lymph system and its structural relationship with other major organ systems of the require further detailed investigations fluke incorporating histochemical, cytochemical and electron microscopical studies. REFERENCES BRAND T. VON 1952. Chemical Physiology of Endoparasitic Animals. Academic Press, New York. CAIN G. D. 1969. Studies on haemoglobins in some digenetic trematodes. Journal of Parasitology 55: 301-306. CHENGT. C. & S-~REISFELD S. D. 1963. Innate phagocytosis in the trematodes Megalodiscus temperatus and Haematoloechus sp. Journal of Morphology 113: 375-380. DUNN T. S.. NIZAMI W. A. & HANNA R. E. B. 1985. Studies on the ultrastructure and histochemistry of the lymph system of three species of amphistome (Trematoda: Digenea), Gigantocotyle explanatum, Gastrothylax crumenifer and
Srivastavaia indica from the Indian Water Buffalo Bubalus bubalis. Journal of Helminthology 59: 1-18. GAIL H. S. 1961. Haemoglobin in trematodes 1. Fasciola gigantica 2. Cotylophoron indiclim. Zeitschrift fiir Parasitenkunde 201 572-575. HAIDER A. S. & SIDDIQI A. H. 1976. Spectrophotometric analysis of haemoglobins of some digenetic trematodes and their hosts. Journal of Helminthology 50: 259-266. JORWN H. E. & REYNOLDSB. D. 1933. The blood cells of the trematode Diplodiscus temperatus. Journal of Morphology 55: 119-130. LEE D. L. & SMITH M. H. 1965. Haemoglobins of parasitic animals. Experimental Parasitology 16: 392-424. Looss A. 1896. Recherches sur la faune parasitaire de
I’Egypte. Premiire partie. Mtmoires de 1’Institut Egyptien. Le Caire 3: 1-252. Looss A. 1902. Uber neue und bekannte Trematoden aus SeeschildkrBten. Zoologische Jahrbiicher. Abteilung fiir Systematik, Okologie und Geographie der Tiere 16: 41 l-
418. LOWE C. Y. 1966. Comparative studies of the lymphatic system of four species of amphistomes. Zeitschrifr fiir Parasitenkunde 27: 169-204. LUTZ P. L. & SIDDIQI A. H. 1967. Comparison of haemoglobins of Fasciola gigantica (Trematoda: Digenea) and its host. Experimental Parasitology 20: 83-87. OZAKI Y. 1937. Studies on the trematode families Gyliauchenidae and Opistholebetidae, with special reference to lymph system. I and II. Journal of Science of Hiroshima University 5: 125-244. OZAKI Y. 1952. Lymph system of Paramphistomum orthocoelium and other two species. Journal of Science of Hiroshima University 13: 79-84. ROHDE K. 1962. Parorientodiscus magnus n.g., n.sp. ein Trematode aus dem Darm von Cyclemys amboinensis (Daud) in Malaya. Zeitschrift fcr Parasitenkunde 21: 457464. ROHDE K. 1963. Orientodiscus fernandoi n.sp. and 0. hendricksoni n.sp. (Trematoda, Paramphistomata) from the intestine of Trionyx spp. in Malaya. Journal of Helminthology 31: 349-358. SHARMAP. N. 1978a. Histochemical localization of succinate dehydrogenase in the lymphatic system of a trematode Ceylonocotyle scoliocoelium. Journal of Helminthology 52: 159-162. SHARMA P. N. 1978b. Histochemical observations on the distribution of monoamine oxidase in the lymphatic system of the Ceylonocotyle scoliocoelium amphistome Fishchoeder 1901 (Trematoda: Digenea). Indian Journalof Experimental Biology 16: 1202-1203. SHARMA P. N. & RATNU L. S. 1982. Morphology, histochemistry and biological significance of the lymphatic system of the trematode Orchocoelium scoliocoelium. Journal of Helminthology 56: 59-67. SMITH M. H. & LEE D. L. 1963. Metabolism of haemoglobin and haematin compounds in Ascaris lumbricoides. Proceedings of the Royal Society, Series B 157: 234-257. STEPHENSON W. 1947. Physiological and histochemical observations on the adult liver fluke Fasciola hepatica L. III. Egg shell formation. Parasitology 38: 128-139.
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system of G. hominis
STRONG P. A. & BOGITSH B. J. 1973. Ultrastructure of the lymph system of the trematode Megalodiscus temperatus. Transactions of the American Microscopical Society 92: 570-578. STUNKARD H. W. 1929. The parasitic worms collected by the American Museum of Natural History Expedition to the Belgian Congo, 190991914. Bulletin of the American Museum of Natural History 58: 233-289. TANDON R. S. 1960a. Studies on the lymphatic system of amphistomes of ruminants: I. Carmyerius spatiosus (Stiles & Goldberger. 1910). Zoologischer Anzeiger 159: 213-217. TANDON R. S. 1960b. Studies on the lymphatic system of
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amphistomes of ruminants: 2. The genera Gastrothylax and Fischoederius. Zoologischer Anzeiger 159:2 17-22 1. THREADGOLDL. T. & ARME C. 1974. Electron microscope studies of Fasciola hepatica XI. Autophagy and parenchymal cell function. Experimental Parasitology 35: 389-405. WILLEY C. H. 1930. Studies on the lymph system of digenetic trematodes. Journal of Morphology and Ph.vsiologySO: l-37. WILLEY C. H. 1935. The excretory system of the trematode, Typhiocoelum cucumerinum, with notes on lymph-like structures in the family Cyclocoelidae. Journal of Morphology 57: 461471.
