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T H E T E G U M E N T O F T H E M O N O G E N E A N G I L L P A R A S I T E S

D A C T Y L O G YR US A M P H I B O T H R I U M A N D D. H E M I A M P H I B O T H R I U M

M. M. Et-NAGoAg* and G. C. KaARN

School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, U.K.'I"

(Received 13 January 1983 )

Abs(ract--Et-N^ooAg M. M. and Kr~g~ G. C. 1983. The tegument of the monogenean gill parasites Dactylog),rus amphibothrium and D. hemiamphibothrium. International Journal for Parasitology 13: 579-592. The general features of the tegument of Dactylogyrus amphibothrium and D. hemlamphi. bothrium, gill p~irasites of the freshwater fish Acerina eernua, resemble those of other monogeneans. However, in D, amphlbothrium the tegument bears spike-like structures associated with tegum~ntal sacs and these have not previously been reported in monogeneans. The origin of the sacs and the possible functions of the spikes and sacs are discussed. It has been found that the tegument covering the two pads located on the ventral surface of D. amphibothrium does not differ in structure from the general body tegument; the pad regions are elevated above the general body surface because of the presence of large glandular sub-tegumentary organs which communicate with the receptaculum seminis. This indicates that the pads play a part in the reproductive biology of the parasite. In spite of the fact that the micro-environments of D. amphibothrium and D. hemiamphibothrium are the same or broadly similar, D. hemiamphibothrium has no spikes, tegumentary sacs or glandular pads and has only two kinds of tegumentary secretory inclusion compared with three kinds in D. amphibothrium. One of the three kinds of cyton in l). atnphibothrium is restricted to the ventro-lateral regions of the posterior half of the body. The tegument of both 13. amphibathrium and D. hemiamphibothrium differs from that of other adult mono- geneans so far investigated in the presence of granular endoplasmic reticulum in the outer syncytial layer.

INDEX KEY WORDS: Dactylogyrus atnphibothrium; D. hemiarapi~ibothrium; monogcneans; gill parasites; Acerina eernua; tegument; ultrastructure.

INTRODUCTION

THE tegument of adult monogeneans has, in general, been found to consist of an outer syncytial region connected via microtubule-lined processes to nucleated regions (cytons) situated beneath the tcgumcntary muscle layers (see reviews by Lyons, 1973; Lumsden, 1975; Rohde, 1975). Only Gyro- dactylus sp., from the skin of Gasterosteus aculeatus, of the monogeneans so far investigated, appears to differ from this general pattern in that the sub- tegumental nucleated regions are said to be absent (Lyons, 1970a). However, Kritsky & Kruidenier (1976) found that cytons are present in Gyrodactylus eucaliae.

Dactylogyrus amphibothrium Wagoner, 1857 and D. hemiamphibothrium Ergens, 1956, from the gills of the ruffe (Acerina cernua L.) were selected for this study for three reasons. First, the only freshwater monogeneans whose tegument has been studied are polystomatids (see Bresciani, 1972; Rohde, 1973) and gyrodactylids (see Lyons, 1970a; Kritsky &

* Present address: Department of Zoology, University o f Mansoura, Mansoura, Egypt.

"1" Address for correspondence.

Kruidenier, 1976). These parasites are in many ways highly specialized monogeneans, polystomatids coping with the special problems of life in the bladder or oral cavity of amphibians or chelonians and gyrodactylids having suppressed the free-swimming larval stage and growing virtually to adult size inside the uterus of the parent. Features of the tegument of these parasites might reflect the specialized nature of their environment. Secondly, preliminary obser- vations revealed the presence of splke-like structures projecting from the body surface of adult specimens of D. amphibothrium. Thirdly, D. amphibothrium possesses a pair of swollen pad-like regions, each of which occupies a ventro-lateral position just anterior to the germarium. It is not apparent with the light microscope whether these pads represent thickened and specialized ventral tegument or whether the sub-tegumentary tissues are involved. For these reasons, it was decided to use electron microscopy to elucidate the structure of the spikes and the pads. The availability of D. hemiamphibothrium together with 19. amphibothrium on the gills of the ruffe offered an opportunity for comparison between the teguments of two related monogeneans occupying micro- habitats which are the same or closely similar.

5"/9

580 M. M, EL-NAGoAn and G, C, KSARN z.,.P, voL. 13. 1983

M AT ERIA LS AN D M ETHODS Each fish was killed by a blow on the head; the gills were

isolated, placed in a shallow dish containing filtered river water and searched with a stereomicroseope. The parasites were dislodged from the host tissue using a fine needle. A silver nitrate staining technique (Lynch, 1933), which is known to reveal positions of surface sensilla, was applied to living unflatt©ned parasites,

The techniques used for preparing ultra.thin (50-70 am) sections for transmission electron microscopy (TEM) and for preparing semi-thin (0.5-1 ~m) toluidine blue stained sections for light microscopy have been described in a previous paper (EI-Naggar & Kearn, 1980).

RESULTS General body tegument of adult Dactylogyrus amphibothrium

The tegument o f adult D. amphibothrium is similar in its basic structure to that of other mono- geneans that have been studied. The tegument is a syncytial cytoplasmic layer connected to cytons lying beneath the tegumentary muscle layers (Fig. 1).

In transverse section the body of D. amphi- bothr/um is oval in outline. The free surface of the outer syncytial layer o f the lateral regions of the body appears to be relatively smooth while the dorsal and ventral surfaces are penetrated by shallow pits (Fig. 2), Both the f ree surface and the inner basal region of the syncytial layer are bounded by a tri-laminate plasma membrane. Small finger-like processes are scattered singly or in small groups over the general body tegument (Fig. 2); each process is covered by the outer plasma membrane and contains a moder- ately electron-dense matrix continuous with that of the syncytial layer, but no secretory inclusions or cytoplasmic organclles have been observed within these processes. A thin filamentous coat is occasion- ally seen external to the surface membrane.

The secretory inclusions found in the outer syn- cytial layer of the tegument of D. amphibothrium appear to be o f three kinds. Secretory bodies Al are relatively large, oval or round in shape and bounded by a single unit membrane (Fig. 2). Although the

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Fro. 1. Diagram show~ng the ultrastructure of the tegument of adult Dacty/ogyrus araphibotRriurn in longitudinal section. Abbreviations: AI, A2, A3, secretory bodies;,bl, basal lamina; bin, basal plasma membrane; e l , C2, C3, cytohs producing AI, A2 and A3 secretory bodies respectivdy; cm, circular muscle layer; co. cytoplasmic connexio~ between cyton and surface tegument; f, finger-like invagination: fp, finger-like process; get, granular endoplasmic reticulum; go, GoJgi body; lm, longitudinal muscle layer; m, mitochondrion; mi, microtubulcs; n, nuflcvs; nu, nucleolus; ore, outer tegumental membrane; s, sac in A2 cyton; sin, basal plasma membrane stack; sp, spike-like structure; ts, tegumental sac; v, membrane bound,

electron.lucent vacuole. Scale bar approximately 2/~m.

LJ.P. vot,, 13. 1983 Tegument of dactylogytids 581

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Fio. 2. Adult D. amphibothrium. The outer tegumental layer. Note that the secretory bodies (Ai) vary in electron ~ensity. p, Shallow pit; other abbreviations as in Fig. l. Scale bar--0.5~m.

FI~. 3. Adult D. amphibothrium. The outer tegumental layer showing Golgi bodies (8o) and granular endoplasmie ~'eficulum (get). Scale b a r - - 0 . 2 5 ~ .

Fzo. 4. Adult 1). amphibothrium. The outer tegumental layer showing a tegumental sac (ts). The sac contains secretorY inclusions (Unlabelled arrows) morphologically similar to A2 secretory bodies, sp, Base of protrudhlg spike. Scale bar = 0.$1an.

Fro. 5. Adult D. amphibothrium. A tegumental spike-like structure (sp) associated with a tegumental sac (ts.) Note that the spike arises from a shallow circular pit (small unlubelled arrows) in the tegument and is enclosed by three membranes

(large unlabelled arrow). Scale bar=0.5~n.

582 M, M. Eh-N^oo^Jt and G. C. K~ARN Id.p. VOL. 13. 1983

majority of A1 bodies have electron-dense contents, some appear to contain electron-lucent material and there are others with contents intermediate in elec- tron density. In the latter, a layer of electron-lucent particles is often observed beneath the surface of the secretory body (Fig. 2). Secretion bodies A2 are smaller than AI bodies and are oval, round, crescent- shaped or irregular in outline (Fig. 2). Each of the A2 bodies is bounded by a single unit membrane and contains moderately electron-dense material. Secretion bodies A3 are membrane-bound vesicles containing a varying number of small electron-dense particles which are embedded in an electron-lucent ground substance (Fig. 2). In many sections the contents of A3 vesicles appear to be released into the tegument matrix. In addition to the three main kinds of secretory inclusion described above, membrane- bound, electron-lucent vacuoles were occasionally observed (Fig. 2) and mitochondria with relativciy few crista¢ are present. The tegument also contains many ribosomes which arc either distributed freely or in groups, glycogen-like structures and a con- siderable amount of granular ¢ndoplasmie reticulum (GER) and Golgl bodies (Fig. 3). Each of the latter consists of a number of membraneous eisternae in association with small, electron-lucent vesicles.

An important feature of the tegument of adult 19. amphibothrium is the presence of relatively large, sac-like structures in the outer syncytial layer (Figs. 1 and 4). These sacs are often found in close prox- imity to the outer tegumental membrane, and each sac is bounded by two unit membranes and contains fine, granular, electron-dense material embedded in an electron-lucent ground substance. The two investing unit membranes appear to enclose between them a homogeneous material with less electron density than the bulk contents. In some sections, secretory inclusions which are morphologically similar to A2 bodies were observed within the tegumental sacs (Fig. 4), There is clear evidence (Figs. zL and 5) that some of these tegumental sacs are associated with projecting spike-like structures, A continuation of the tegumental sac penetrates the base of the spike and provides the core of each spike. Consequently each spike is enclosed by three mem- branes (Fig. 5), the outer tegumental membrane and beneath it the two membranes enclosing the tegu- mental sac. No desmosomes were observed between the outer tegumentai membrane and the outer membrane of the sac, but the spike arises from a shallow circular pit in the tegument and the outer tegumental membrane lining this pit comes into intimate contact with the outer membrane of the tegumental sac, forming a structure resembling a tight junction {Fig. 5). In the region of the root of the spike and in the base of the spike the layer of homogeneous material between the inner and outer sac membr'.mes is considerably thicker. In sections, this layer is seen to separate the tegumental sac contents and the sac material in the core of the spike.

It is possible that there may be ~ narrow connecting passage between the tegumentai sac contents and the core of the spike but such a passage has not been found. Those spikes bear no resemblance internally to eili¢, and, in spite of an intensive search, no nerve eonnexions have been found associated with either the tegumental sacs or with the spikes.

Finger-like invaginations of the tegumental basal plasma membrane which are filled with basal lamina material occasionally intrude (rite the outer syncytial layer and in addition to these single invaginations, basal plasma membrane stacks, each comprising usually 2-6 parallel membraneous cisternae, are also present (Fig. 1). In some places, cisternae of each stack lie parallel to and close to the inner surface of the basal plasma membrane, while in other places, cisternae of each stack lie perpendicular to the basal plasma membrane (Fig. 1). The stacks are sometimes found to be associated with the f'mger-like invagi- nations of the basal plasma membrane mentioned above. Neither the finger-like invaginations nor the stacks have been seen to traverse completely the tegument matrix. Hemi-desmosomes have not been found in association with the basal plasma mem- brane where it makes contact with the basal lamina. The latter is continuous with a layer of fibrous inter- stitial material which is variable in thickness and surrounds the cytons. Two layers of tegumental muscle fibres are present, an outer circular and an inner longitudinal muscle layer, and the mito- chondria distributed among the muscle fibres are large with numerous relatively long cristae and are more abundant than those found in the outer syncytiai layer.

Three kinds of eyton were recognized in adult D. amphibothrium (see Fig. 1). The cytons which produce AJ secretory bodies are restricted in their distribution. They are located in two groups, one situated in each ventro-lateral region of the posterior half of the body. Cytoplasmic connexions between A1 cytons and the outer syncytiat layer are lined by microtubules but few such connexions have been found. Cytons producing A2 secretory bodies and those producing A3 vesicles are 'distributed all over the body, although A2 cytons appear to be more abundant in the lateral regions of the body. Many microtubule-lined connexions were observed between A2 cytons and the outer syncytial layer but so far no conncxions have been found between A3 cytons and the surface tegument.

Each AI cyton is muir(nucleate and in a single section as many as six nuclei were encountered. The nuclei are irregularly shaped and contain grahular nueleoplasm with a ,relatively large nucleolus positioned centrally (Fig. 6). The outer membra~ae of the nuclear envelope is studded with ribosomes. The cytoplasm of the A I cyton is highly electron-dense and packed withextensive, dilated a n d narrow cisternae of GER. While the narrow cisternae of the GER are filled with an electron-lucent material, each

l.J,v, voL. 13. 1983 Tegument of dactylogyrids 583

of the dilated cisteraae possesses a.central core of moderately electron-dense material embedded in an electron-lucent ground substance. Cisternae of smooth ¢ndoplasmic reticulum are present in the cytoplasm but they are less abundant than those of the GER, Golgi bodies' are numerous and present in all regions oi" the cytoplasm; their cisternae and the associated vesicles contain an electron'lucent sub- stance. In the vicinity of Golgi complexes, large, oval to round, AI secretorY inclusions with moderately electron-dense contents were observed but away from Golgi regions and particularly in the connexions between the cytons and thc surface tegument many of the AI inclusions have highly electron-dense con- tents and appear smaller in size than the corre- sponding inclusions found in the vicinity of Golgi complexes. Aggregations of A1 secretory bodies are sometimes found (Fig: 7) but more observations will be necessary to determine whether these aggregations lie close to or within the proximal end of the cyto- plasmic connexion between the cyton and the surf~/ce tegument, A few mitochondria were observed in the A1 cytoplasm.

The cytoplasm of A2 cytons is moderately elec- tron-dense when compared with that of AI cytons and there is clear evidence that some A2 cytons, perhaps all of them, are multinucleate (Fig. 8). Each nucleus has a single nucleolus and contains small chromatin patches which are often condensed at the periphery of the nucleus. The nuclear envelope is studded externally with many ribosomes and pene- trated by many nuclear pores. The cytoplasm of A2 cytons is invested by a layer of fibrous interstitial material which, in some places, appears to be associated with muscle fibres (Fig. 8). Ribosomes are abundant and widespread; cisternae of GER are also present but they are more abundant at the periphery of the cytoplasm. Each Golgi body is composed of 6-12 flattened membraneous cisternae tog&her with associated small vesicles (Fig. 8, inset). These cisternae, which may be straight or curved, contain electron-dense material while associated Golgi vesicles appear to have electron-lucent contents. Mitochondfia and glycogen-like components are also present.

In addition to A2 secretory bodies, the A2 cyto- plasm has been found to contain structures invested by two membranes, mbrphologically similar to the tegumental sacs found in the outer syncytial layer except that they are smaller in size (Fig. 9). These structures were not observed in AI cytons.

Unlike AI and A2 cytons, A3 cytons are uni- nucleate (Fig. 10). Each cyton has a single, irregularly shaped nucleus with a conspicuous, centrally situated nucleolus. The cytoplasm of A3 cytons is highly electron-dense and this appears to be consistent with the observation that free ribosomes constitute quantitatively the most abundant cell organeUe. Golgi complexes are numerous, each consisting of a number of membraneous cisternae which contain an

electron-lucent substance. These cisternae were often found to be surrounded by a large number of A3 vesicles. A small number of mitochondria containing a few cristae are also present.

General body tegument of young D. amphibothrium The general body tegument of immature specimens

of D, umpMbothrium collected from the gills (specimens in which the copulatory organ is not completely formed) was found to be iimilar to that of the adult worm. The outer syncytial layer contains the same kinds of secretory inclusion (AI, A2 and A3) as in the outer syncytial layer of the adult. Three corresponding kinds of cyton were identified and their distribution appeared to be similar to that in the adult parasite. The outer syncytial layer of immature parasites contains numerous ribosomes and many mitochondria. Golgi bodies and cisternae of GER are present but the impression was gained that they are less abundant than in the outer syncytiai layer of adults, Sac-like structures were found also in the outer syncytial layer of immature specimens but they are less numerous and enclosed by a single unit membrane. The .sacs contain small, electron-dense particles embedded in an electron-lucent ground substance (Fig. 12). Structures similar to these sacs were occasionally observed in the A2 cytoplasm. No spikes have been found so far on the outer surface of the immature parasite.

The ventral pads of D. amphibothrium Staining with silver nitrate failed to reveal any

sensilla on the surface of the ventral pads of D. amphibothrium although sensilla were abundant elsewhere. Resin-embedded, toluidine blue stained sections and TEM sections revealed that the tegument covering the pads is not modified and that the pad regions are elevated above the general bQdy surface because of the presence of large glandular organs beneath the tegument (Figs. 11) 13). R~in- embedded sections stained with toiuidine blue have revealed that the inner borders of the two glandular organs communicate with each other in the mid-line of the body and that a single, narrow process extends posteriorly from this junction. TEM sections show that this process penetrates the epithelium lining the receptaculum scminis (Fig. 14). No microtubules have been observed to line the terminal portion of the process and no desmosomal junctions have been found between the terminal portion of the process and the adjacent epithelium lining the receptaculum seminis.

The glandular organ is bounded by a thin, dense, basal plasma membrane which, at numerous points, protrudes into the cytoplasm of the glandular organ in the form of. finger-like invaginations (Fig. !1). Some.invaginations are longer than others and they contain material that is continuous with the under- lying basal lamina. Beneath the basal lamina, separate groups of muscle fibres are found.

584 M. M. EL-N^ooAa and G. C, i~ARN

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hJ.P. VOL, 13. 1983 Tegument of dactylogyrids

0 0 F~G. 1 I. Diagram showing the uhraslructure of one of the two pads of D. amphibolhrium, av, Aggregation of electron-lucent vesicles; c, central cell; cp, connexion with the pad on the other side of the body; gp, glandular processes in the tegument; mf, muscle fibres~ ms, large mass of electron-dense material; psc, peripheral sync~tial cytoplasm; sb, electron-dense secretory bodies; t, tegument. Other abbreviations as

in Fig. 1. Scale bar approximately 2pro.

Two kinds of cell were recognized in the pads, gland ceils lying at the periphery which constitute the major part of the organ (Fig. 13) and another kind o f cell found in the central region of the pad (Fig. 19). The evidence suggests that the cytoplasm of the peripheral gland cells is syncytial and that the inner cytoplasm of these cells is thrown into many compli- cated folds (Figs. 11, 16). Where adjacent folds come into contact, the opposing plasma membranes are separated from each other by a narrow, uniform, extracellular gap (Fig. 17) and desmosomal or other specialized intercellular connexions have not been observed. A charact©ristic feature of the peripheral glandular syncytium is that most o f the nuclei are located near the surface o f the organ. The peripheral g land cell cytoplasm is highly electron-dense and contains a considerable amount of GER, the cisternae o f which run parallel with each other and

with the plasma membrane (Fig. 18). Golgi bodies are distributed throughout the cytoplasm, each com- prising 3-5 flattened eisternae in association with small vesicles and both the cisternae and the vesicles are filled with electron-dense contents (Fig. 18). Free ribosomes and a few mitochondria are also present. Electron-dense bodies which are either oval or circular in outline are abundant in the cytoplasm, particularly near the surface o f the organ (Fig. 13). In addit ion, large masses of electron-dense material (Fig. 16) as well as aggregations of electron-lucent vesicles (Figs. 11, 15) were frequently observed.

At the region o f the glandular organ near the external body surface of the parasite, the peripheral syncytial cytoplasm sends out numerous processes which penetrate the outer tegumental layer (Figs. I l , 13). These processes are sheathed by the basal lamina which exhibits continuity with that enclosing the rest

Fla. 6. Adult D. arnphibot!wium. Cyton producing AI secretory bodies. Note that each of the dilated clsternae of GER (unlabelled arrows) posses~ a central core of moderately electron-dense material embedded in an electron-lucent ground

substance, ger, Granular endoplasmic reticulum; go, Goigi body; n, nucleus; nu, nucleolus. Scale bar = l~m. FIG. 7. Adult D. ampkibothrium. Aggregation of AI bodies surrounded by cytoplasm of At cyton.:Scal© bar=~m. Fio. 8. Adult D. amphibotkrium. A2 cyton showing that the cytoplasm is multlnucleate. A2, small, membrane-bound, moderately electron-dense bodies; 1, fibrous interstitial layer; m f, muscle fibre; n, nucleus; nu, nucleolus. Scale bar = 1 ~m.

Inset: Golgi body in cytoplasm of A2 cyton. Scale bar=0.25~m. Fla. 9. Adult D. amphibothrium. Sac (s) enclosed by two membranes lying in cytoplasm of A2 cyton. Scale bar = 0.5~m. FIo. 10. Adult D. amphibothri~m. A3 cyton producing A3 vesicles, go, Golgi hods; n, nucleus; nu, nucleolus. Scale bar= Ij~m.

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LJ.P. VOL. 13. 1983 Tegument of dactylogyrids 587

of the organ. The processes are packed with the electron-dense bodies characterizing the peripheral syncytial cytoplasm. No desmosomal junctions have been observed betw~n these processes and the surrounding tegumental layer and no evidence ~as been found that their contents are discharged in'T['~t he outer tegumental layer or released outside the body. The tegument covering the pads closely resembles that covering the general body surface except that the outer surface of the tegument covering the pads appears to be more folded than elsewhere (Fig. 13). No cytons or microtubule-lined processes w e r e found between the pads and the tegument.

The central region of each pad contains another kind of cell which is strikingly different from the peripheral glandular region. Only one cell has so far been observed but it is possible that more than one of these cells may occur. The cell has a single nucleus and the cytoplasm, which is not folded, is low in electron density and contains GER which is usually located at the periphery of the cell (Figs. 11, 19). The cisternae of GER are circular in profile and enclose fine granular material with an electron density similar to that of the ground substance of the cyto- plasm. The cytoplasm of the central cell contains mitochondria, ribosomes and oval to round, moderately dectron-dense bodies. No desmosomal or other intercellular connexions have been observed between the plasma membrane of the peripheral glandular syncytium and that of the central cell.

General body tegument of adult D. hemiamphi- bothrium

The general body tegument of D. hemiamphi. bothrium resembles that of D, amphibothrium, There are pits in the outer syncytial layer hut in D. hemiamphibothrium they penetrate deeply into the basal region of the layer (Figs. 20, 21). The outer syncytial layer in D. bemiamphibothrium contains numerous, randomly distributed mitochondria and free ribosomes. GER and Golgi bodies were observed but they are less abundant than those found in the outer syncytial layer of D. amphibothrium. Two kinds of secretory inclusion were identified: type H1

bodies are oval to round in profile and contain electron-dense contents whereas type H2 inclusions are membrane-bound vesicles containing small electron-dens¢ particles (Fig. 21). No tegumental sacs, projecting spikes or glandular pads have been foundtin D. hemiamphibothrium,

Tht cytons in D. hemiamphibothrium are of two kinds, ts~r,~roducing HI bodies and the other pro- ducing H2 vesicles. There is evidence that many, perhaps all, of the HI cytons are multinucleate. Each nucleus is oval to round in profile and possesses a single, conspicuous nucleolus positioned centrally (Fig, 22), The nucleus contains a small number of chromatin patches which are located peripherally. The cytoplasm of HI cytoas is not homogeneous;'some areas are electron-dense, packed with ribosomes and containing elaborately developed Golgi bodies surrounded by dilated cisternae of GER, whilst !n other areas the cytoplasm appears comparatively pale, owing to the fact that few ribo- somes are present. These electron-lucent areas appear to be free of GER and they may represent areas of badly preserved glycogen similar to the areas des- cribed in Amphibdellaflavolineata by Lyons (1971).

H2 cytons are uninueleate. The cytoplasm is highly el~tron-dense and contains Golgi bodies, mito- chondria, free ribosomes and GER (Fig. 23).

Regional specializations of the tegument The tegument lining the anterior adhesive sacs of

both species of dactylogyrid monogenean has been described in detail elsewhere (El-Naggar & Kearn, 1980) and the tegument of the haptor has not yet been studied. o The tegumen t lining the anterior region of the buccal cavity of D. amphibothrium and D. hemiamphibothrium has no characteristic features differentiating it from that ,:peering the rest of the body. In D. amphibothrium, the tegument lining the anterior region of the buccM cavity has numerous tegumental sacs and spikes ere present. However, the tegument lining the posterior region of ~ the buccal cavity differs from that covering the rest of the body and will be described elsewhere.

FiG. 12. Immature 1). umphibothr/um. Outer ~yncytial layer ~owing a tegumental sac (ts) containing finely granular, eloctron- dense particles and enclosed by a single unit membrane. A1, tegumental secretory inclusion. Scale bar = 0.5/~m.

FiG. 13. Adult D.'amphibothriura. Peripheral syncytial cytoplasm of the glandular pad which sends out numerous processes (gp) into the tegument (t). The processes are packed with electron-dense secretory bodies. Scale bar=4#m.

FiG. 14. Adult D. araphibothrium. Inner process (ip) of the glandular pads at the poim where it penetrates (unlabclIed arrow) ~e ~ithelium (e) lining the receptacnlum scminis (rs). sb, Electron-dense secretory bodies. Scale bar= l~m.

FIG. ]~L A~,alt D. amphibothriura. Aggregation of electron-lucent vesicles in the peripheral syno/tial cytoplasm of the glandular pad. Scale bar = 0.5/~m.

Fro. 16. Adult D. amphibothrium. Glandular pad showing that the inner cytoplasm of the peripheral gland cells is thrown into many folds, ms, Large mass of electron-dense material; n, nucleus; nu, nucleolus; sb, electron-dense secretory bodies.

Scale bar= tern. Fro. 17. Adult D. amphfbothrium. The peripheral cytoplasm of the glandular pad. Note that the opposing plasma membranes (mdabelled a;rows) are separated by a narrow uniform extraedlular gap. sb, ~ n . d c m e secretory bodies. Scale bar=0.Sjan. FIG. 18. A~lt D. ampl~ibothrium. The 1-e:ripherat cytoplasm of the glandular pad showing the Golgi body (go) and granular

e~doplasmic L-eticnlum in the peripheral cytoplasm. Scale bar---lbtm. Fro. 19. Adult D. amphibothriura. Central cell cytoplasm of the glandular pad. get, Granular endoplasmic reticulum; rob,

moderately electron-dense body. Scale bar=0.Spm.

5 8 8 , M. M. EL-N^ooAR and G. C. K~^R~ I,|,P. VOL, 13. 1983

FiG. 20. Diagram showing the ultrastructure of the tegument of adult D. hemiamphibothrium in longitudinal section. C1, C2, cytons producing HI and H2 ~ecretory bodies respectively; el, electron-lucent area; p,

tegumental pit; r, ribosomes. Other abbreviations as in Fig. 1. Scale bar approximately Ipm.

DISCUSSION The tegument of both Dactylogyrus amphi-

bothrium and D. hemiamphibothrium was found to be a syncytial cytoplasmic layer communicating with cytons lying beneath the tegumentary muscle layers. In this respect, the tegument of D. amphi- bothriura and D. hemiamphibothrium resembles that of other monogeneans. However, the tegumental spike-like structures associated with tegumental sacs in D. amphibothrium have not been reported pre- viously. The two pads located on the ventral surface of D. amphibothrium also appear to be a unique feature. It has been found that the tegument covering the pads does not differ markedly in structure from the general body tegument; the pad regions are elevated above the body surface because o f the presence of large, subtegumentary, glandular organs. The inner borders of these organs communi- cate with each other in the mid-fine of the body and from this mid-point o f communication a short process penetrates the epithelium fining the recep- taculum seminis.

A further discovery concerns the distribution of the cytons in D. amphibothrium; evidence hasl been found to indicate that one of the three kinds of

cyton (A l) is restricted to the ventro-lateral regions of the posterior half of the body. In addition, the tegument of both D. amphibothrium and D. hemiarnphibothrium differs from that ot" other adult monogeneans so far investigated in the presence of GER in the outer syncytial layer, Ttte apparent absence of surf~e microvilli, except on the specialized tegument linin~ the anterior adhesive sacs (see EI-Naggar & Kearfi, 1980), is shazed with Polystomoides spp. (see Rohd¢, 1973, 1975) find with Rajonchocotyle (see Lyons, 1972) but mierovi]li have been reported on the surfaces of many other mono- geneans.

In D. amphibothrium the tegumental sacs contain fine, granular, electron:dense material embedded in an electron-lucent ground substance and each sac in the adult is surrounded by two unit membranes. The cytoplasm of A2" cytons occasionally contains structures resembling the tegumental .~acs, except that the A2 sacs are smaller. Furthermore, the tegumental sacs were found t o contain secretory inclusions mo~'phologically"similar tO' A2 secretory bodies. I t is' possible,'therefor.e, that' the tegumental sacs are manufactured in the A2 ¢y[ons and then dischaxged, lik~e other tegumental secretions, into

t.J,P, voL, 13. 1983 Tcgumo~t of dactylogyrids 589

Fie. 21. Adult D. hemiamphibothrium, The outer ~umenta l layer showing the two kinds of secretory inclusion (I-If and H2). Notcthat the surface pits (p) pcn¢tratv dc~ly into the t©lumcnt: mr, Muscl© fibre.

Scale bar = l~m.

Fie. 22. Adult D. hemiamphibothrlum. H I cyton showing the electron-lucent areas of the cytoplasm (el). gcr, Dilatvd cistcrnac of sranulax endoplasmi¢ rcticulum; So, Golgi body; n, Nucleus, Scale b a r : 1 ~ .

Fro. 23. Adult D. hemiamphibothrium. H2 cyton. Note that the cytoplasm is highly electron-dense except in the region' where H2 vesicles axe found, t, TegumenL Scale barffi l/~nn,

590 M. M. EL-N^GGAg and G. C. K~^aN tJ.p. VOL. 13. 1983

the outer syncytial layer through the cytoplasmic connexions. However, there is also a similarity between the contents of the sacs and the contents of A3 tegumentary secretory vesicles suggesting that A3 vesicles may contribute to the sacs.

Sac-like tegumentary inclusions have occasionally been described in other platyhelminths. There is a resemblance between the tegumentary sacs found in juvenile specimens of D. amphibothrium and 'reservoirs' enclosed by a single membrane and con- taining granular material found by Dorsey (1975) in the cytoplasm of the preacetabular gland of schisto- some cercariae. Bulbous structures containing numerous vesicles were reported by Wittrock (1978) in the syncytial tegument covering the ventral papilla of the digenean Quinqueserialis quinque- serialis. These bulbs were presumed to be sensory structures although they were not shown to have connexions with the nervous system. Dixon & Mercer (1965) reported a bulb-like structure associated with a fine, non-ciliary tegumentai projection near the oral sucker of the cercaria of Fasciola hepatica. This bulb was found to contain mitochondria, synaptic vesicles and neurofilaments and a connexion was found with a nerve trunk. It seems unlikely that the spikes and tegumental sacs of adult D. amphi- bothrium act as sensory receptors since no trace has been found of nerve connexions to them.

There are references to the presence of bristles and spines on the tegument of monogeneans. Morris & Halton (1971) observed small bristle-bearing protuberances on the tegument lining the mouth region and covering the buceal suckers of Dicli- dophora merlangi. They showed that the tegumental matrix beneath these regions is dense and fibrillar in appearance and they suggested that the projecting bristles might have an abrasive or adhesive function. In D. amphibothriurn some spikes and numerous sacs have been observed in the tegument lining the anterior region of the buccal cavity but they appear morphologically and structurally different from the bristles described by Morris & Halton.

Paling (1966), in his light microscope study of the attachment of Diplectanum aequans, noted the presence of scales covering the posterior part of the body in the region just anterior to the haptor and suggested that scales might be involved in attach- ment. Oliver (1976), in a scanning electron micro- scope study of D. aequans found spines projecting from the surface of the tegument in the posterior region "~f the body. These spines were shown by Shaw (1980) to consist of a thin layer of electron- dense, sclerite material lying beneath the outer tegumental membrane. Kritsky (unpublished Ph.D thesis, University of Illinois, 1971) found that the cirrus spines in Gyrodactylus eucaliae were manu- factured in and remained embedded in the tegu- mental cytoplasm. These surf~Ice scales or spines are restricted in their distribution to a particular region of the body, while in D. amphibothrium spike-like

structures were found all over the body surface. Furthermore, there is no resemblance between the internal ultrastructure of the spikes of D. atnphi. bothrium and that of the scales or spines described by Shaw and by Kritsky.

it is interesting to note that spikes were not observed in immature specimens of D. atnphi- bothrium and although tegumenta[ sacs are present in young specimens, they appear to differ from those of the adult. This suggests that the function of the spikes and sacs might he related to sexual maturity. One possibility is that the spike con- tents, which appear to b¢ derived from the sacs, may diffuse through the wall of the spike providing chemical clues permitting parasites to identify sexually mature individuals of their own species. Such identification might involve contact ehemo- perception (the surfaces of adult parasites are well- endowed with projecting cilia which are presumed to be sense organs). There is also a possibility that a chemical trail may be established by pheromones diffusing out of the spikes into the surrounding water. It is noteworthy that sacs and spikes appear to be absent in D. hemiamphibothrium which shares the gills of ruffe with D. amphibothrium and it would be of interest to know whether similar organs occur in other dactylogyrid gill parasites.

The glandular pads of D. amphibothrium com- prise a peripheral syncytium producing oval to round, electron-dense bodies and a central cell with a paler cytoplasm containing occasional moderately electron-dense bodies. In addition to the electron- dense bodies in the peripheral syncytial cytoplasm, large masses of highly electron-dense material have also been observed; these masses may originate by coalescence of the smaller dectron-dense bodies. The tegument covering the pads contains the same inclusions as the rest of the general body tegument and since there is no room for cytons in the region of the glandular organ, it seems likely that these inclusions are transported from eytons located out- side the region of the pads.

The role of the pads in the biology of D. amphi. bothrium is as enigmatic as that of the spikes and tegumental sacs. Since the inner portion of the p,~zls makes intimate contaet with the receptaculum seminis, it seems likely that the glandular pads play a part in the reproductive biology of the parasite. In view of the presence of abundant secretory bodies in the outer pad cytoplasm and the penetration of glandular processes into the tegument, it is tempting to suggest that the secretion has a pheromonal function, but the glandular processes do not appear to reach the surface of the tegument and no secretory bodies from the glandular pads have been observed in the tegumental cytoplasm. A sensory function for the pads is unlikely since no specialized surface features sucii as sensilla were found.

The outer region of the adult tegument of .9. amphibothrium and D. hemiamphibothrium has

l.J.p, r o t . 13. 1983 Tegument of dactylogyrids 591

been found to contain a moderate amount of GER, often associated with Golgi bodies and free ribo- somes. GER has not previously been reported in the adult tegument of monogeneans but does occur in the embryonic tegument of Gyrodactytu~ eucaliae according to Kritsky & Kruidenier (1976). The presence of ribosomes in the tegument was reported by Shaw (1980) in Diplectanum aequans. Some of these were associated with unique epidermal scales (see above) and Shaw suggested that these ribosomes may be involved with synthesis of scale material, However, in larger individuals he also reported the presence of groups of ribosomes elsewhere in the tegument covering the body and haptor, a phenom- enon which suggests changes in tegument activity and function as the parasite ages, Ribosomes were also reported in the tegument of Rajonchocoryle emarginata by Lyons (I 972).

Golgi bodies have been encountered more fre- quently in the monogenean tegument. They have been found by Morris & Halton (1971) in Dicli- dophora merlangi, by Lyons (1970a) associated with lamellate bodies in Gyrodact¥1us sp., by Lyons 0972) associated with the ribosomes mentioned above in R. emarginata and by Shaw (1980) in the haptor tegument of D. aequa~.

The presence of GER, ribosomes and Golgi in the tegument of adult specimens of D. amphibothrium and D. hemiamphibothrium is of particular interest. This suggests that ~ynthetic activity is not restricted to the cytons; perhaps the electron-lucent vacuoles occasionally encountered in the tegument of D. amphibothrium are manufactured by these organelles.

Three kinds of cyton (AI, A2 and A3) have been found in D. amphibothrium, while only two kinds (H1 and H2) were observed in D, hemiamphi- bothrium. In each species, each kind of cyton supplies the tegument with secretory bodies which are morphologically distinct from those produced by the other cytons. Morris & Halton (1971) suggested that two morphologically distinct secretory inclu- sions are manufactured in a single cyton in Dicli- dophora merlangi and this also seems to occur in Gyrodactylus eucaliae (see Kritsky & Kruidenier, 1976). Lyons (1970b) reported only one kind of secretory inclusion in the tegument of the skin parasite Entobdella soleae.

The differences between D. amphibothrium and D. hemiamphioothrium in the number of secretory inclusions and in other features of the tegument are unlikely to be related to environmental differences since the two parasites inhabit the gills of the ruffe and their micro-environments are likely to be the same or closely similar.

All the three kinds of cyton in D. amphibothrium were observed in the posterior half of the body, while only two (A2 and A3 cytons) were observed in the anterior half of the body. In spite of this arrange- ment, all the three kinds of secretory inclusion are

distributed throughout the outer syncytial layer all over the body. Kritsky & Kruidenier (1976) refer to a similar restriction of cytons to the dorsal half of the posterior part of the body in G. eucaliae, but the two kinds of secretory inclusion were widespread in the surface syncytium. Rees & Day (1976) studied the origin and development of the tegument in the cercaria of Cryptocoeyle lingua, They reported that the secretory inclusions discharged from the eytons lying ventrally, immediately in front of the excretory vesicle, were found only in the ventral tegument corresponding with the position of their cytons. In D. amphibothrium it might be supposed that A1 cytons have disappeared from the anterior region of the adult parasites after shedding their secretion into the anterior tegument, but this is not borne out by observations on immature gill parasites in which AI cytons also appear to be restricted to the posterior half of the body. Thus, the evidence suggests that AI secretory inclusions are manufactured in cytons in the posterior half of the body and then transported to the tegument covering the anterior half of the body, but how this transport is brought about is not known.

In the cytoplasm of AI cytons, the membrane- bound secretion bodies are variable in appearance. In the vicinity of Golgi complexes, these inclusions appear oval to round in profile with fibrillar, moderately electron-dense contents. As soon as they move from Golgi regions, particularly in the cyto- plasmic conneTdons between the cytons and the surface tegument, many of these inclusions become more electron-dense. It appears that the diameter of the newly formed AI secretory bodies is slightly larger than that of the older ones. This may indicate that the contents of these secretory bodies undergo condensation while moving towards the outer syncytial layer.

Little is known about the chemical nature and possible functions of tegumental secretory inclusions in monogeneans. Lyons (1970b, 1971) suggested that the dense bodies in the outer tegumemal layer of E. aoleae, Acanthocotyle elegan~ and Amphibdella flavolineata may be mucoproteinaceous because they gave a slight positive diastase.fast reaction with the PAS test. She found that the tegumentary inclusions of E. soleae showed/1-metachromasia with toluidine blue) a phenomenon which is said to be a property of mucoproteins and she sug$csted that the mucus coat may be derived from the tegumentary inclu- sions. Kritsky & Kruidenier (1976) presented evidence to indicate that one of the two secretory inclusions in the tegument of G. eucaliae expels its contents at the tegumental surface but no such evidence has been found in D. amphibothrium or in D. hemiamphi- bothrium. However, there are indications that A3 vesicles in D. amphiboehrium may release their contents into the tegument matrix.

In both D. amphibothrium and D. hemiamphi- bothrium, the basal plasma membrane intrudes into

592 M. M. EL-N^~Aa and G, C. Ke^~tN

the outer syncytiai layer of the tegument and these intrusions take the form of either finger,like invaginations or membrane stacks. Similar structures have been observed in the tegument of almost all the monogeneans so far described, However, in P/ectanocotyle gurnardt, the parallel folds of the basal plasma membrane extend through almost the entire thickness of the tegument (Lyons, 1972). Also, in Pot.vstoma integerrimum some invaginations of the basal plasma membrane extend as far as the distal surface of the tegument and are intimately associated with mitochondria (Bresciani, 1972). Because of the close similarity between the basal invag[nations in Polystoma and those found in cells specialized for water or ion transport, Bresciani suggested a similar function for the basal invagi- nations in the tegument of P. inlegerrimum. In D. amphlbothrium and D. hemiamphibothrium neither the finger-like invaginations nor the stacks have been seen to traverse the tegument matrix completely or to be associated with mitochondria. It is possible that these invaginations and stacks act as a means of attaching the thick tegumental layer to the basal lamina, as already suggested by Lyons (1969) for the basal infoldings of the tegument of Gyroeotyle urns.

Acknowledgements--We arc most grateful to Mr. N. Baker for help with electron microscopy, to Mr. G. Cleveland for technical assistance and to Mr. M. Hardy for photographic advice. We are indebted to the Anglian Water Authority and particularly to Mr. K. Jones, Dr. J. Wortley, Mr. D. Leafy and the late Mr. D, Pye for providing fishes during the course of this investigation. The work was conducted during the tenure of an Egyptian Government Scholarship by the senior author.

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