Ultrastructural and cytochemical studies on the ventral pouch of Gastrothylax crumenifer (Digenea: Paramphistomidae)

ULTRASTRUCTURAL AND CYTOCHEMICAL STUDIES ON THE VENTRAL POUCH OF GASTROTHYLAX CRUMENIFER (DIGENEA : PARAMPHISTOMIDAE) T. S. DuNN,$# R. E. B. HANNA* and W. A. NIZAMI~ * Zoology Department, t Zoology

Department,

The Queen’s University of Belfast, Belfast, BT7 lNN, Northern Ireland Aligarh Muslim University, Aligarh 202 001, Uttar Pradesh, India.

(Received 27 August 1985; in revisedform 2 October 1986) HANNA R. E. B. and NIZAMI W. A. 1987. Ultrastructural and cytochemical studies on the ventral pouch of Gastrothylax crumenifer (Digenea:Paramphistomidae). International Journalfor Parasitology 17: 1163-l 173. The ventral pouch of G. crumenifer consists of an extensive atrium, opening just behind the mouth and ending blindly at the level of the testes. It is lined by a thin tegument underlain by numerous flask shaped pyriform cells. The non-glandular nature of the pyriform cell cytoplasm and the presence of haemoglobin and numerous mitochondria suggest that the pouch fulfils primarily a respiratory role. Contractions and relaxations of musculature associated with the pouch may ventilate the pouch with ruminal fluid, or with gaseous oxygen which may be present in small quantities in the rumen. Oxygen taken up by haemoglobin in pyriform cells may be transferred to haemoglobin in the lymph system, and thence distributed internally.

Abstract-DUNN T. S.,

INDEX KEY WORDS: Gastrothylax crumenifer; paramphistome; microanalysis; histochemistry; haemoglobin; respiration.

INTRODUCTION THE

ventral

pouch;

ultrastructure;

X-ray

Existing accounts give only brief descriptions of the ventral pouch, usually little more than details of its size and shape. The present study provides the first ultrastructural description of this system as it occurs in Gastrothylax crumenifer.

are thick-bodied, conical or cylindrical digeneans, distinguished from other forms by the possession of a posteriorly located acetabulum. The more familiar species are parasites of domesticated livestock, especially in the tropics and subtropics. Members of the family Gastrothylacinae, which includes the genera Gastrothylax, Fischoederius , Carmyerius, Wellmanias and Johnsonitrema, are all parasites of the forestomach of large ruminants. They are characterized by the possession of an extensive, tegument lined atrium occupying almost the entire ventral half of the body. This structure, commonly referred to as the ventral pouch, shows few differences in general morphology between the various species. It has a single opening anteriorly, close to the mouth, and posteriorly it ends about the level of the testes. Its precise function is unresolved, however, in the majority of pouched paramphistomes the genital pore opens anteriorly into the ventral pouch, which has led to speculation that the pouch represents an enlarged genital atrium (Nasmark, 1937) and may function as a brood chamber for eggs (Innes, 1912). paramphistomes

MATERIALS AND METHODS Mature specimens of G. crumenifer were collected

$ Present address: Zoology Department, University of Oklahoma, 730 Van Vleet Oval, Norman, OK 73019, U.S.A. 1163

from buffaloes, Bubalus bubalis, slaughtered in Aligarh, India. Specimens were washed briefly in Hanks BSS and processed for light microscopy and transmission (TEM) and scanning (SEM) electron microscopy. For general histochemical tests (Table 1) parasites were fixed in 10% neutral phosphate buffered formaldehyde (PBF), washed in buffer, dehydrated with ethanol, embedded in Paraplast and sections cut at 12 pm. For demonstration of lipids, PBF-fixed specimens were embedded in Tissue-Tek II OCT Compound (Lab-Tek Products, Naperville, Illinois) supercooled for 30 s in Analar hexane at -70°C. Cryostat sections (12 lrn) were collected on coverslips and stained with Sudan Black B. For light microscopic enzyme histochemistry, fresh specimens or specimens prefixed for 1 h at 4’C in-double distilled glutaraldehvde in 0.05 M sodium cacodvlate buffer (pH 7.41, were embedded in Tissue-Tek OCT and further processed as outlined above. Sections of fixed material were stained for acid phosphatases or non-specific esterases. Fresh frozen sections were stained for succinate dehydrogenase or adenosine triphosphatase. For TEM, sliced specimens were fixed for 8 h at 22’C in 4% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, subsequently washed in buffer and post-fixed for 1 h in 1% OsO,, dehydrated in ethanol and embedded in Durcupan

Xylene and absolute alcohol pretreatment Substrate omitted Mouse liver

Substrate omitted

Mouse liver

Lipids-phospho; neutral and non-acidic Acid phosphatases

Non-specific esterases

Succinate dehydrogenase

Adenosine triphosphate

Sudan black B (Bayliss & Adams, 1972)

Note:

+f+

intense activity; $+ moderate activity; + mild activity; 0 no activity.

Nitrotetrazolium blue (Chayen, Bitenzky, Butcher & Poulter, 1969). Lead nitrate (Wachstein, Meisel& Niedzwiedz, 1960)

Fresh @men sections

Azo dye (Burstone, IYSN) Indoxyl acetate (Holt & Withers, 1952)

Pre-fixedfrozen sections

Ferric iron

Ped’s Prussian blue (Bancroft CLStevens, 1977)

-

+

+/0

0

-+

++

0

0

+

0

-

0

i-f/+++

0

+I’++

+/o

0

++

0

-

0

0

0

+++

0

+

0

0

-

Nuclei

-

+

i-t-+

++/++-ibasal 0

+-+

I-

++

+

-

+/o

_

+

0

-

0

-

-

t/o

+/o

0

Control

Pyriform cells

Nuclei

-k+

-t/O

++

+/o

+i-

-I-+

c/o

++

+/o

++

Boiling water for 30 min Boiling water for 30 min -

Test

i-t+

t+

+

ff

Amylase treated at 3’7°Cfor 1 h

Control

-+-

Test

Control

Tegumental cells

Test

Control

G. crumenifer

Tegumental syncytium

TESTS ON THE VENTRAI. POUCH OF

1, Blood smear 2. Incubated at 130°C for 30 min BIood smear

RNA

DNA

General proteins

General carbohydrates and amylase-resistant carbohydrates General proteins

Test for

I-HISTOCHEMICAL

Haemoglobin

Mercuric bromophenol blue (Pearse. 1960 Coomassie brilliant blue Methyl green (Pearse, 1960) Pyronin Y (Pearse, 1960) Pickworth’s benzidine method (Gurr, 1958)

Periodic acid/Schift’s (Liliie, 1951)

Wax sections

Method

TABLE

Ventral

pouch of G. crumenifer

resin. Sections were double stained with many1 acetate and lead citrate and examined using a Jeol 1OOCX electron microscope. For SEM, parasites were washed thoroughly in Hanks BSS, fixed in 4% aqueous glutaraldehyde, washed in cacodylate buffer and post-fixed in 1% 0~0,. Specimens were subsequently dehydrated in acetone, critically dried, gold coated and examined using a Jeol35CF SEM. For X-ray microanalysis, parasites were cut into four or six pieces using a glass knife and fixed for 4 h in 1% double distilled glutaraldehyde buffered with 0.1 M cacodylate buffer, pH 7.4. Some specimens were subsequently washed in buffer, dehydrated in acetone and embedded in Spurr resin. Thick sections (250 nm) were mounted on aluminium grids and then coated with carbon and examined using a Jeol 1OOCX TEMSCAN analytical microscope at 100 kV (probe diameter 0.1 pm). Other specimen pieces were buffer washed, dehydrated in ethanol, critically dried and mounted on carbon stubs. These bulk specimens were then coated with carbon and examined using a Jeol Superprobe 733 analytical SEM at 15 kV (probe diameter 10 ,um). For the ultrastructural localization of acid phosphatases specimens were fixed 15 min at 4°C in 1% doublehistilled elutaraldehvde buffered with cacodvlate, PH 7.4. Fixed specimens were sliced (approx. 50 pm) and ieplaced in the fixative for a further 15 min. Buffer washed slices were incubated in 100 ml of 0.05 M acetate buffer (pH 5.0) containing 0.2 g lead citrate plus 10 ml of 0.1 M sodium pglycerophosphate for 40 min at 37°C. Sodium ,!?glycerophosphate was omitted from control incubations. Slices were then washed briefly in distilled water and further fixed for 2 h at 4°C (as above), buffer washed, dehydrated with ethanol and embedded in Spurr resin. RESULTS

Light microscopic observations The ventral pouch of G. crumenifer comprised an

extensive atrium occupying approximately a third of the body volume (Fig. 1). A small aperture to the pouch occurred posterio-ventral to the mouth. A short, narrow ‘neck’ led from the aperture to the pouch. The pouch often appeared triangular in transverse section, the apex directed dorsally. The aperture of the ventral pouch was observed to open and close periodically in live specimens, particularly when a rumen was freshly opened and worms exposed to air. Specimens placed in saline after removal from the rumen frequently floated. The ventral pouch of fresh cut worms displayed an intense red coloration. The ventral pouch was lined by the tegumental syncytium. Elongate, flask shaped cells, hitherto referred to as the pyriform cells, were located directly beneath the outer syncytium (Fig. 2). Pyriform cells beneath ventral portions of the pouch generally were larger (approx. 70 pm long and 6 pm wide at the base) than those beneath the dorsal surface (approx. 30 ,~rn long and 5 ,~rn wide), while only a few small cells occurred around the neck of the pouch (Fig. 1). Pyriform cells were readily distinguishable from the surrounding parenchyma in sections stained with PA/S and Orange G (Fig. 2); the pyriform cells avidly incorporated Orange G while the parenchyma gave an intense PA/S reaction. Sections stained for haemo-

1165

globin revealed intense reactions within the pyriform cells and lymph vessels, while the parenchyma was unstained (Fig. 4). Other histochemical properties of the pouch are presented in Table 1. Ultrastructural observations

The oral and ventral pouch openings were surrounded by a series of concentric ridges, bearing numerous boss-like sensory papillae (Fig. 3). The ultrastructure of these papillae has been described by Dunn, Hanna & Nizami (in preparation). Papillae also occurred in the neck of the pouch, however, they differed in that ciliated nerve bulbs within these papillae were usually associated with a bulbous extension of the tegumental syncytium (Fig. 8). The remainder of the ventral pouch was devoid of papillae, however, non-ciliated sensory bodies were found in the base of the tegumental syncytium (Fig. 10). The tegument lining the pouch also was folded into numerous transversely arranged ridges and furrows (Figs. 2, 4, 5). High magnification SEM showed that the tegumental surface was elevated by tightly packed rounded microvilli (Fig. 6). TEM revealed the tegument of the pouch was 23 pm thick (Figs. 7,9, 10, 1 l), compared to the outer body tegument at approx. 17 ,um. However, the morphology was similar (Dunn, Hamra & Nizami, in preparation). Both types of tegumental secretory inclusions, denoted Tl and T2 bodies, were present although T2 bodies were much less common in the pouch (Fig. 11). Separate Tl and T2 tegumental cells were located between the pyriform cells. Mitochondria were absent from the tegument of the pouch, as they were from the exterior tegument. There was no evidence for pinocytosis at the apical membrane. The pyriform cells were located directly beneath the distal tegumental cytoplasm and were separated from it by a thin basal lamina and layer of interstitial material (Fig. 11). The distal cytoplasm of each cell was elevated into numerous finger-like projections (1-2~~ long and O.l-0.2ym wide), which protruded into the base of the tegument, but at no time penetrated the basal membrane (Figs. 7,9, 11). Tight junctions and desmosomes occasionally occurred between the two systems. The cytoplasmic matrix of the pyriform cells was finely granular or homogenous and exceedingly electron dense. Characteristically the cytoplasm contained numerous elongate mitochondria (Figs. 7, 9) with extensive, longitudinally arranged cristae (on average 10 per mitochondria; Fig. 13). The matrices of mitochondria were variable and generally depended upon location within the cell: apical mitochondria were dense (Fig. 12); mid region mitochondria showed various degrees of electron opacity and, at times, vacuolation (Fig. 13) with occasional electron dense vesicles (Fig. 16); basal mitochondria were more vacuolated, so that in some cases the integrity of the mitochondria appeared disrupted (Figs. 14, 17). Also within the basal cytoplasm were electron dense

1166

T. S. DUNN, R. E. B. HANNA and W. A. NIZAMI

Ventral

pouch of G. crumenifer

bodies, morphologically resembling tertiary lysosomes (Figs. 7,9,15, 17). Generally, they were about the same size and shape as mitochondria, but some were small and elongate while others were large and irregularly shaped. Each was bounded by a double membrane. The matrix usually contained a number of regularly aligned membranes and electron lucid or opaque vacuoles (Fig. 15), possibly representing lipid. Considering the morphological similarity of these bodies with mitochondria in the pyriform cells and the presence of acid phosphatases (AcPase) in their matrices (Fig. 1S), as well as in electron dense vesicles resembling primary lysosomes in the surrounding cytoplasm, it was presumed that these bodies represented autolysed mitochondria which remained as tertiary lysosomes. Other inclusions of the pyriform cells included glycogen granules, electron lucid vesicles (Figs. 9, 15) and myelin figures. Each cell possessed a single basally located nucleus (Fig. 9). Granular endoplasmic reticulum (GER) and Golgi bodies were not observed. The basal membrane of the pyriform cells formed tight junctions with lymph and excretory vessels and parenchyma. Frequently spheroidal protrusions of pyriform cells extended into the peripheral cytoplasm of the parenchyma. These protrusions usually were devoid of mitochondria or residual bodies and resembled lymph (Dunn, Nizami & Harma, 1985) (Fig. 17). Isolated membrane bound areas of cytoplasm of similar appearance, also were found in the base of pyriform cells suggesting protrusion of lymph vessels into these cells (Fig. 12). Set amongst the pyriform cells were occasional ramifications of nervous tissues and small cells containing numerous electron dense vesicles, presumably representing neurosecretory material. The musculature associated with the ventral pouch consisted of transverse fibres inserted into the interstitial material beneath the tegumental syncytium and extending between the pyriform cells and parenchyma to the exterior tegument; three or four blocks of circular muscle situated between the basal portions of the pyrifotm cells; a small band of oblique muscle beneath the circular blocks; and a large discontinuous layer of longitudinal muscle beneath the pyriform cells. FIG. 1. Light photomicrograph,

sagittal section Ap, aperture

1167

TEM localization of AcPase in the ventral pouch revealed concentrated activity associated with the distal invaginations of the pyriform cells into the tegumental syncytium. Most deposits were found on the pyriform cell side of this interface. Other sites of activity were the lateral and basal plasma membranes of pyriform cells, particularly at tight junctions, and tertiary lysosomes (autolysed mitochondria) and electron dense vesicles within the cytoplasm (Fig. 18). The apical cytoplasm and apical plasma membrane of the tegumental syncytium also showed reactivity. Spectrum analysis of thick sections revealed a predominance of iron within the pyriform cell cytoplasm (Fig. 19). Microanalysis of bulk specimens revealed relatively low concentrations of iron throughout the parasites, however, significantly higher concentrations were evident in the pyriform cells (average 6.64%), as compared with the parenchyma (average 0.2%) or areas beneath the external body tegument (average 0.11%) (Fig. 20, Table 2). Some globular structures situated between ‘parenchymal spaces’ yielded exceptionally high concentrations of iron (up to 45%). However, the exact nature of these structures and their relationship with surrounding tissues could not be determined. Consistently high concentrations of silicon (up to 42% of all elements analysed) were registered within the ‘parenchymal spaces’ and beneath the ventral pouch. Other main elements present are detailed in Table 2. DISCUSSION

Little information exists concerning the ventral pouch apart from details of the size and shape in the various species (Fischoeder, 1903; Stiles & Goldberger, 1910; Maplestone, 1923; Fukui, 1929; N&mark, 1937). Consequently, the functional significance of the pouch remains unresolved. Innes (19 12) amd N&mark (1937) assumed the ventral pouch to be an enlarged genital atrium, however, it is difficult to envisage such a derivation for the ventral pouch of Carmyerius exoporus and Johnsonotrema magnum, in which the genital pore is situated on the exterior surface. Innes (1912) also suggested that the pouch may function as a brood chamber for ova. However, in G. crumenifer eggs are rarely found within the lumen of the pouch.

of G. crumenifer stained with PA/S and Orange to pouch; VP, ventral pouch; Te, testis.

FIG. 2. Light photomicrograph, sagittal section of ventral pouch in G. crumenifer, stained bar = 25 pm). Cm, circular muscle; Om, oblique muscle; Par, parenchyma; PC, pyriform Tm, transverse muscle. anterior

of G. crumenifer (scale bar = 50,~m). arrows, sensory papillae.

Ap, aperture

G (scale bar = 1 mm).

with PA/S and Orange G (scale cells; T, tegumental syncytium;

FIG. 3. SEM

micrograph,

to ventral

FIG. 4. Light

photomicrograph, sagittal section of ventral pouch in G. crumenifer, stained bar = 100 ,u m). LV, lymph vessels; Par, parenchyma; PC, pyriform cells.

pouch;

for haemoglobin

FIG. 5. SEM micrograph, sag&ally bissected specimen of G. crumenifer (scale bar = 2 mm). AC, acetabulum; to ventral pouch, Ph, pharynx; Vp, ventral pouch; arrow, ‘neck’ of ventral pouch. FIG. 6. SEM micrograph,

tegumental

Op. mouth; (scale

Ap, aperture

surface of ventral pouch in G. crumenijer (scale bar = 0.5 pm).

1168

T. S. DUNN, R. E. B. HANNA and W. A. NIZAMI

Ventral pouch of G. crumenifer

Ultrastructural observations on the ventral pouch of G. crumenifer indicate a close morphological resemblance to the ventral papillae and ridges of various notocotylid digeneans, as described by Beverley-Burton & Logan (1976) MacKinnon (1980, PhD thesis, University of Aberdeen, Scotland, 1982a), Wittrock (1978) and Radlett (1980). The papillae and ridges similarly are covered by a thin syncytial tegument, underlain by large, non-glandular pyriform cells containing numerous elongate mitochondria. This morphological similarity suggests a functional similarity. Various functions have been attributed to the ventral papillae of notocotylids, including adhesion (Beverley-Burton & Logan, 1976; Radlett, 1980), extracorporeal digestion (Harwood, 1939) nutrient absorption (Wittrock, 1978) and absorption and metabolism of oxygen (MacKimron, 1980, PhD thesis as cited above, 1982c). It is difficult to envisage an adhesive role for the ventral pouch, considering that all pouched forms have a large acetabulum. Similarly, present observations on the pouch seem to be inconsistent with roles in extracorporeal digestion or nutrient uptake. Although the pyriform cells display an intense protein reaction, this does not appear to be associated with glandular activity, and probably can be related with ultrastructural proteins or haemoglobin. There is also a lack of secretory bodies within the pyriform cell cytoplasm, and an absence of Golgi bodies and GER, which are normally associated with cells engaged in biosynthesis. Furthermore, the tegument of the ventral pouch shows little modification for absorptive purposes. It is devoid of microvilli and lamellae which generally characterize absorptive surfaces in helminths (Wittrock, 1978; Erasmus, 1972) and there is no evidence for macromolecular uptake by pinocytosis. However, both ATPase and AcPase, which have been implicated,,in tegumental absorptive and secretory processes (Ohman, 1966; Fujino, Threadgold & Ishii, 1983) were localized in the pouch suggesting that the tegument may be capable of absorbing certain micromolecules. MacKinnon (1982a, b) suggested that the primary function of the ventral papillae of Notocotylus trheriah was in the absorption and metabolism of oxygen. This deduction was based on the presence of numerous mitochondria, haemoglobin and a number

1169

of respiratory enzymes within the pyriform cells of the ventral papillae. In view of the fact that many aspects of histology, histochemistry and ultrastructure are shared by the papillae of notocotylids and the ventral pouch of G. crumenifer, it is proposed that the latter structure functions as a respiratory chamber. The pyriform cells of the ventral pouch contained numerous elongate mitochondria each with a complex series of septate cristae, indicative of high metabolic activity (Crompton & Joyner, 1980). Mitochondria in other cell types were smaller and contained fewer cristae, while in some tissue systems, such as the tegument and lymph, they were apparently absent. Possibly mitochondria are concentrated in the pyriform cells in order to take advantage of any oxygen which may be present in the ventral pouch. Assuming that the requisite enzymes are present to permit aerobic metabolism, excess ATP generated in these cells might be distributed to other cell types less well positioned to engage in aerobic respiration. MacKinnon (1982c) identified a number of respiratory enzymes in the pyriform cells of N. triserialis, and demonstrated the dependence of this parasite on aerobic respiration in cyanide inhibition experiments. Succinate dehydrogenase (SDH) was the only respiratory enzyme demonstrated within the pyriform cells of G. crumenifer. Although it is an important enzyme in the Krebs cycle it also operates in the anaerobic fumarate reductase system (Kurelec, 1975). The functional significance of the numerous autolysed mitochondria in the basal portions of the pyriform cells in G. crumenifer is unclear but possibly there is a high turnover of mitochondria within these cells. Lysosomal degradation of mitochondria was not observed by MacKinnon (1982a) in the pyifotm cells of N. triserialis, “membrane bound membrane” in the of N. attenuatus

however, Radlett (1980) found vesicles containing loose scrolls of basal portions of the pytiform cells [= N. triserialis]. These could rep-

resent tertiary lysosomes or myelin bodies. MacKinnon (1982a) proposed a mechanism for the eversion and retraction of the ventral papillae of N. triserialis in response to oxygen demand. Everted papillae are held in close aposition to the host gut mucosa. Clearly the ventral pouch of G. crumenifer is not capable of eversion but it may undergo periodic contractions to produce an aerating current of

Frc. 7. TEM micrograph, tegumental syncytium (T) and pyriform cells (PC) of the ventral pouch in G. crumenifer (scale bar = 10 pm). Cm, circular muscle; F, finger-like projections of pyriform cells; M, mitochondria; Ns, nervous tissue; Tm, transverse muscle; T,, tertiary lysosomes. FIG. 8. TEM micrograph, ciliated sensory nerve ending in a tegumental papilla, G. crumenifer (scale bar = 2 y m). Bb, basal body; C, cilium; D, ring desmosome; Ds, dense rings; Nb, nerve bulbs; T, tegument; TX,bulbous appendage of tegument. FIG. 9. TEM micrograph, G. crumeni$zr. Pyriform cells (scale bar = 5 pm). (Abbreviations as in Fig. 7.) Cm, circular muscle; V, vacuoles. FIG. 10. TEM micrograph, G. crumenifer. Un-ciliated sensory nerve endings in the tegumentary syncytium (T) (scale bar = 0.5 ,u m). D, ring desmosome; Ds, dense rings; Rt, rootlet. FIG. 11. TEM micrograph, G. crumenifer. Tegumentary syncytium of the pouch (T). (Scale bar = 1.Opm) Bl, basal lamina; F, finger-like projections of pyriform cells; It, interstitial material; PC, pyriform cell; Tb, tubercles.

1170

T. S. DUNN, R. E. B.

HANNA

and W. A. NIZAMI

Ventral

1171

pouch of G. crumenifer SilK”l

CulK4

Al lK4

0

KeV

10

FIG. 19. X-ray emission spectrum from thick sections of pyriform cell cytoplasm in G. crumenifer showing Ku (6.403 keV) and KB (7.057 keV) peaks for iron.Extraneous peaks for aluminium (1.487 keV) and copper (8.047 keV) are derived from support grid and components of the electron microscope, respectively. 0

ruminal fluid through

the pouch.

Tegumental

structures in the neck of the pouch may monitor and control these respiratory currents (i.e. rheoreceptors). The non-ciliate sensory receptors, located in the tegument of the pouch, may function as mechano- or proprio-receptors and possibly control the degree of dilation of the pouch. Levels of oxygen within the rumen generally are low (approx. 2 mm Hg; von Brand, 1952) however, levels may periodically increase with intake of fodder or water. Conceivably, gaseous oxygen entering the rumen may be taken up into the ventral pouch by ‘mouthing movements’, similar to those observed in live, freshly exposed specimens in situ. Interestingly, G. crumemfer shows a predilection to locate in the dorsal aspects of the various chambers of the rnmen where gases might become trapped (Dunn, PhD thesis, Queen’s University, Belfast). The thinness of the tegnmental syncytium lining the ventral pouch and elaboration of the basal plasma membrane may serve to increase the rate and efficiency of oxygen uptake from the lumen of the pouch into the pyriform cells. Similarly, tight junctions between the syncytium and the pyriform cell membranes and pyriform cells and lymph or parenchyma sensory

FIG. 12. TEM micrograph, FIG. 13. TEM

micrograph,

FIG. 14. TEM

micrograph,

G. crumenifer.

FIG. 15. TEM

micrograph,

G. crumeltijer

FIG. 16. TEM micrograph, FIG. 17. TEM

micrograph,

FIG. 18. TEM micrograph,

FIG. 20. X-ray emission spectrum of pyriform cells in bulk specimens of G. crumenifer, showing Ku and K/3 peaks for iron and K/3 peaks for silicon, ahuninium and potassium.

TABLE 2-PERCENTAGECOMPOS~TION OFTHEMAIN ELEMENTS IN CERTAIN TISSUES OF G. CrurneniferAs DETERMINED BY XRAY MlCROANALYSlSOFBULKSPEclMENS

Percentage

Element

Pyriform

cells

composition

Parenchyma

External tegument

Silicon Aluminium Magnesium Sodium Potassium Iron Sulphur

11.49 3.45 4.10 5.35 1.14 6.64 0.80

25.03 1.30 2.50 2.60 0.31 0.20

0.28 0.17 0.30 0.90 0.41 0.11 0.95

Total analysed

32.91

31.94

3.12

G. crumenifer. Mitochondria (M) in the apical cytoplasm LV, lymph vessels. G. crumenifer.

10

KeV

ciliated

Mitochondria (M) in the apical/mid (scalebar = 1.0 pm). Mitochondria (M) in the bar = 1 .O ,Um). V, vacuole.

of pyriform portions

mid/basal

(scale bar = 0.5 pm). Gy, glycogen; arrow, cristae-like membranes.

of cytoplasm

regions T,,

(scale bar = 1.0 pm). LV, lymph T,, tertiary lysosome.

G. crumenifer.

Localization

of acid phosphatases 1.0 ,um).

lysosome; fused primary

vessel; Par, parenchyma; in tertiary

of pyriform

of pyriform

tertiary

G. crumenifer. Dense bodies (arrowed) in mitochondria may represent or possibly inorganic salt deposits (scale bar = 0.5 ,u m). G. crumengeer

cells (scale bar = 1 .O pm).

lysosomes

cells

(scale

V, vacuole; lysosomes

PC, pyriform (arrows)

cells

cell;

(scale bar =

1172

T. S: DUNN, R. E. B. HANNA and W. A. NI~AMI

may represent sites for inward diffusion of oxygen and/or ATP and other small metabolites (Gallagher & Threadgold, 1967; Matricon-Gondran, 1980). Thus, oxygen may be taken up from the ventral pouch lumen by haemoglobin in the pyriform cells, then transferred to haemoglobin in the lymph system (Dunn, Nizami & Hanna, 1985) and delivered to various internal tissues. Likewise, excess ATP produced in the pyriform cells may be distributed to other cell systems. High concentrations of silicon, as recorded from the tissues beneath the ventral pouch and between the parenchymal ‘spaces’, have not been described previously from tissues of digeneans (Halton, 1982a, b). Conceivably, silicon could be incorporated into such tissues as interstitial material in order to impart structural rigidity, possibly for more efficient muscular action. Alternatively, a high silicon content in tissues might be a protective strategy for survival within the abrasive environment of the rumen. Acknowledgements-This work was completed whilst T. S. D. was in receipt of studentships from the Department of Education for Northern Ireland and The Queen’s University of Belfast. Technical assistance rendered by Dr. Mohammad Ahmad and Mr. Abbas Abidi (AMU) during field collections and by the personnel of the Central EM Unit (QUB) is gratefully acknowledged.

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pouch of G. crurnenifer

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