On the Anatomy of Cercaria Macrocerca from Sphaerium Corneum.

Filippi (1854) first described a Cercaria macrocerca, but Ssinitsin (1907) showed that at least four species of cer-cariae were included in Filippi’s original description. Of these four species, Ssinitsin’s Cercaria gorgoderae pagen-stecherihad previously been described by Thiry (1860) under the name of Cercaria macrocerca (Fit).

The name Cercaria macrocerca was retained by Lube (1909) for a cercaria which Ssinitsin claimed to be the larva of Gorgoderina vitellilobae (Olss.), a parasite in the bladder of frogs.

Sewell (1922), using the relative measurements of the tail as diagnostic characters, separates the macrocercous cercariae into a ‘Gorgodera’ group and a ‘Gorgoderina’ group. In both groups the tail is very large, and its proximal part forms a chamber in which the body is completely enclosed ; the members of the ‘Gorgoderina ‘group are distinguished by the fact that the proximal third of the tail is distended into a large swollen globe, sharply marked off from the distal narrow stem.

The cercaria described below corresponds, in most respects, with Lube’s (1909) description of Cercaria macrocerca (Fil), and falls into Sewell’s ‘Gorgoderina’ group.

This work was undertaken with the intention of providing a more complete description of a gorgoderine cercaria and sporocyst than the inadequate accounts existing; particular attention has been paid to the characteristic cells lining the excretory vesicle, since the cytological characters and the function of these cells have never been investigated.

This work has been carried out in the laboratory of Professor H. Graham Cannon, to whom I am indebted for much technical advice.

The study was suggested by Mr. F. J. Brown, to whom it is difficult to express adequately my gratitude for his continued advice and assistance

Specimens of Sphaerium corneum Linn, collected at Pickmere, in Cheshire, were isolated in small glass vessels. The mature cercariae liberated by infected molluscs were studied alive.

All measurements were made on living material subjected to moderate pressure under a No. 1 cover-glass.

As a mounting medium fresh ox serum was found extremely useful, since it both stimulates the flame-cells and has a clearing effect on the tissues ; neutral red proved to be the most useful vital stain.

Individual cercariae and whole molluscs containing sporocysts were sectioned. The best fixative for histological purposes was found to be ‘Susa’ warmed to about 50° C., whilst for cytological purposes ‘Flemming’s, without acetic’, was used. Sections were stained in iron haematoxylin and counter-stained with orange G ; for demonstrating the stylet glands and cysto-genous glands Mallory’s triple stain was very satisfactory.

The sporocyst of Cercaria macrocerca is situated between the gill lamellae of Sphaerium corneum together with developing mollusc embryos.

The reproduction of lightly parasitized molluscs is not entirely prevented, but in a heavy infestation more than a hundred sporocysts may be present, entirely filling and distending the space between the gills; in such cases the pressure the sporocysts exert is sufficient to prevent the development of mollusc embryos.

The gill lamellae are ruptured by the cercariae which are continually escaping; in this way lesions are caused and large areas of the gills may be put out of action.

The sporocyst is a white, club-shaped sac, attached by its narrower end to the inner surface of the gill. When full grown it measures up to 2 mm. long by 0·7 mm. broad at the wider end, and contains about a dozen cercariae.

There is no birth pore and no recognizable pharynx.

The surface of the sporocyst is covered by a wrinkled cuticle, under which lie a circular layer and a longitudinal layer of muscle-fibres.

The young sporocyst shows considerable power of movement ; but, as it becomes distended by the developing cercariae, the cuticle becomes stretched, the distances between the parallel muscle-fibres greatly increase, and the sporocyst remains motionless.

Beneath the muscle is a layer, one cell thick, and composed of two types of cells. The majority of the cells are large and cubical, with vacuolated cytoplasm and with round nuclei in which the nucleolus is small.

Scattered amongst these cells are smaller ovoid muscle-cells, connected to the circular and longitudinal fibres, and having large, deeply staining, oval nuclei surrounded by a small amount of somewhat granular, deeply staining cytoplasm.

The interior of the young sporocyst is filled with large, vacuolated, parenchymatous cells, amongst which the germballs develop. As the germ-balls increase in size, they come to lie in a cavity at the distended free end of the sporocyst, the narrower attached end still being packed with parenchymatous cells, amongst which a few very young germ-balls are found.

On each side of the sporocyst is a wide, twisted, main excretory canal, which is joined by an anterior and a posterior lateral canal, each of which collects from five flame-cells. Each sporocyst thus contains twenty flame-cells, which are placed amongst the parenchyma.

The flame-cell is of a peculiar type figured by Thiry (1860) and by Looss (1894). By a study of material fixed in ‘Flem-ming’s without acetic ‘and sectioned at 4 μ I am enabled to amplify previous descriptions.

The flat sheet of fused cilia comprising the vibratile ‘flame’ (Text-fig. 2, v.f.) is greatly expanded laterally in older cells, and is contained within a chamber the walls of which are strengthened by numerous annular bands of deeply staining material (Text-fig. 2, s.b.).

Quemer (1929) noted that a single strengthening band is present in the small type of flame-cell such as is more usual in adult trematodes. The flame-cell of the cercaria is of this type (Text-fig. 3).

The deeply staining nucleus of the cell lies close up against the basal plate of the cilia, and the whole is surrounded by a thin layer of cytoplasm which sends out processes to the adjoining cells.

As the sporocyst ages, the flame-cells increase in size. In the youngest cells observed the sheet of vibratile cilia was approximately in the shape of an equilateral triangle, whose base measured 0-013 mm. The cells in full-grown sporocysts have the base of the triangle greatly lengthened (up to 0·053 mm.) and curled inwards at each end. At the same time the excretory ducts are widened, and in this way a much greater volume of fluid can be dealt with by the excretory apparatus without increase in the number of flame-cells.

Free-swimming cercariae (fig. 1, Pl. 11) are liberated from infected molluscs at any period during day or night. Twenty infected molluscs were isolated in separate glass vessels and kept for 23 days in darkness at a temperature of 18° C., the water being changed daily. The largest number of cercariae produced by any one Sphaerium in a single 24-hour-period was 145; the same mollusc, 9 mm. long, liberated 1,276 cercariae during the 23 days it was kept under observation.

It was found that cercariae lived in tap-water for about 22 hours, in 0-75 per cent NaCl Solution for 30 hours, and in sterile ox serum for at least 60 hours, the temperatures in each case approximating to 17° C. ; at a lower temperature (12° C.) the length of life was increased at least 50 per cent., and above 20° C. the length of life was considerably decreased.

The tail is divided into two regions, a proximal swollen globe, 0·42 mm. by 0.25 mm. and an elongated distal stem, 0·8 mm. by 0·12 mm.

The whole larva is covered by a transparent elastic cuticle ; at the proximal end of the tail-globe this is raised into a circular fold completely surrounding and closing over the body of the cercaria, and forming the so-called ‘chamber’ with a minute anterior pore (fig. 1, Pl. 11).

The ventral lip of the excretory pore on the body is extended to provide a socket in which a small knob, at the proximal extremity of the tail, is enclosed.

The cuticle is continuous over body and tail, and the only other connexion between the two consists of muscle-fibres which radiate from the tail knob to the posterior walls of the body. In iron haematoxylin stained sections the aggregation of fibres in the tail knob appears as a solid dark mass, from which a band of muscles passes down each side of a short stalk (fig. 1, st., Pl. 11) through the globe of the tail, and continues as a double band down each side of the stem. Conspicuous ovoid muscle-cell bodies with large nuclei are placed along these longitudinal muscle-bands, being especially numerous in the stalk connecting with the body, and at the point where the stem dilates into the globe.

Throughout the tail, circular muscle-fibres lie beneath the cuticle, and external to the longitudinal muscles ; typical muscular cell-bodies are connected with them on the inner side, and each muscle-cell is connected to those adjacent by fine protoplasmic threads.

At the proximal end of the globe are about a dozen very large cells with clear cytoplasm and round nuclei (fig. 1, gl. 1, Pl. 11). These cells, which have been referred to as ‘glands’ or as ‘packing cells’ by various authors, stain blue with Mallory’s triple stain in the same way as mucus cells, to which they are very similar in appearance.

Another small group of non-muscular cells is located between the longitudinal muscle-bands at the point where the globe narrows into the stem (fig. 1, gl. 2, PI. 11). These cells are small, with a small amount of clear cytoplasm which in life has a very faint greenish tinge, and from them a strand, running down the centre of the stem, appears to arise. This strand (fig. 1, c.str., Pl. 11) is a chain of small elongated cells with oval nuclei, and ends at the extreme tip of the tail. Fine protoplasmic threads pass from these cells to the muscle-cells. Thiry (1860) and Wesenberg-Lund (1934) refer to this chain of cells as an excretory canal. It is certainly not a canal, and does not appear to be muscular, since it does not contract, it does not stain deeply with haematoxylin as do the muscle-fibres, and its cell-bodies are quite unlike those of the muscles.

The extreme tip of the tail is covered with minute spines and is drawn in by muscular action to function as a sucker by means of which the animal attaches itself to the substratum (Text-fig. 4). The longitudinal muscle-bands of each side of the tail contract alternately, causing the free end (tail-globe and ‘chamber’) to sway rapidly to and fro.

The cercaria does not swim voluntarily, and always remains close to the mollusc from which it was liberated, nor does the body seek to emerge from the tail chamber unless moribund or subjected to unnatural conditions ; in such cases the fore body is bent ventralwards, thus bringing the dorsally placed points of the stylet (fig. 3, Pl. 11) to bear on the enclosing cuticular walls, which can then be ruptured by scratching with the stylet.

Ssinitsin (1907) states that the cercaria is swallowed by carnivorous insect larvae (Epitheca, Agrión) ‘… la cercaire pénètre dans le corps de l’hôte intermediaire d’une façon passive, en lui servant de proie. Ainsi la chambre protectrice sert au Distomide de kyste temporaire, et le protège contre l’action masticatrice des mandibules d’insecte’.

On one occasion I have obtained cysts of Cercaria macro-cerca from larvae of Chironomous pedellus (De G.) which had been kept in contact with cercariae for a fortnight. The cysts, two or three in each larva, were situated at the anterior end of the insect amongst the muscles. Further attempts to obtain cysts in aquatic insect larvae have not so far been successful, so that it is impossible to evaluate Ssinitsin’s statements.

(b) Anatomy of the Body (figs. 4 and 5, PI. 12).

The body of the cercaria may easily be squeezed out from the tail chamber under moderate pressure of a cover-glass.

It is an elongated oval, 0·54 mm. by 0·12 mm., when at rest, but it may be extended to 0·78 mm. long or contracted to 0·31 mm. long.

The stylet gland-cells (see p. 321) and the excretory vesicle cells have a distinct brownish colour.

The oral sucker measures 0·085 mm. long by 0·08 mm. broad, and is somewhat smaller than the ventral sucker (0·096 mm. long by 0·092 mm. broad).

A thick cuticle covers the body; minute alternating ridges and grooves across the cuticle cause it, in surface view, to appear finely striated transversely.

A stylet (fig. 3, Pl. 11) 0-017 mm. by 0-0065 mm., of peculiar form, is embedded in the oral sucker dorsally to the mouth; its long axis is dorso-ventral to the body, and it has three points which are directed slightly backwards. The central point is the largest and is borne on a keel which extends to the base of the stylet. The upper edge of the keel is attached along a tube which is semicircular in cross-section and closed at the anterior end, which bears the two small lateral points. Along each side of the keel, towards its lower edge, is a narrow blunt blade.

Haematoxylin and acid fuchsin are taken up readily by the stylet in fixed specimens.

Small conical spines line the mouth cavity and the acetabulum, the latter being fringed by longer needle-shaped spines. The cuticle is otherwise unarmed, but down each side of the body is an irregularly double row of sensory papillae of the type figured by Bettendorf (1897). These are most numerous anteriorly ; six of the papillae are set around the opening of the ventral sucker.

—Immediately beneath the cuticle are three muscle-layers, such as are usually described in adult Trematodes, and are described in Cercaria vitrina by Neuhaus (1936).

In the fore-body are many dorso-ventral fibres. A strong group of these radiates from the region of the future genital opening to the dorsal surface of the body. Similar fibres radiate from the ventral sucker to the body-wall.

—The oral sucker surrounds the mouth, which leads into a sinuous oesophagus lined by an outer circular layer and an inner longitudinal layer of muscles. There is no pharynx. The gut bifurcates at a point about one-sixth of the body length behind the oral sucker; the intestinal branches, of irregular diameter, pass backwards on either side of the ventral sucker, and reach almost to the posterior extremity of the body. No musculature has been observed in the intestinal branches, whose walls are composed of a mosaic of flattened cells with small nuclei and clear cytoplasm.

—A wide commissure, passing dorsally over the oesophagus, links together the two lateral ganglia, from each of which (fig. 4, n.g., Pl. 12) four main nerve-trunks arise. Of the two anterior trunks, the outer supplies the bodywall and periphery of the oral sucker, the inner vanishing into the posterior end of the sucker.

A lateral nerve passes out to the body-wall, where it divides into anterior and posterior lateral branches.

The posterior nerve from each ganglion is a thick trunk passing down the body laterally to the ventral sucker; small nerves to the cuticular sensory papillae can be seen branching out from the two posterior trunks, which are joined together by a narrow commissure just in front of the ventral sucker.

Nuclei of the nerve-cells are closely packed around the fibrillar ganglion masses.

Genital Rudiment (figs. 4 and 5, Pl. 12).—The genital system of the cercaria is already well developed.

Just behind the ventral sucker, on the right of the body, is the spherical ovary; like the testes, it is formed of closely packed cells, having large nuclei and a small amount of clear cytoplasm.

Laurer’s canal opens by a small dorsal pore on the left of the body, and connects with a duct arising from the ovary. This duct then turns sharply forwards, a small lobe of the vitelline gland joining it on each side, and continues dorsally over the ventral sucker as the uterus, which joins with the large ventrally placed seminal vesicle, just behind the bifurcation of the gut.

Of the two ovoid testes, one is usually placed on the right, beside the excretory vesicle, the other on the left, in front of the excretory vesicle. In living cercariae the fine vasa deferentia may be seen arising from the testes, but their junction and further course have not been observed.

Looss (1894) gives an excellent figure of the reproductive system of a Cercaria macrocerca which is probably identical with the cercaria here described. But he describes all the organs as being on the reverse sides of the body. Of twenty-five cercariae liberated from the same mollusc on one day, it was found that four had the ovary on the left and Laurer’s canal on the right, whilst the remainder had the more normal arrangement. Further investigation showed that about 15 per cent, of cercariae have the ‘reversed’ arrangement, and that both types of cercaria may occur in one and the same sporocyst.

Stylet glands (figs. 4 and 5, g.c., PL 12).—Twelve large gland-cells fie dorsally in the body posteriorly to the bifurcation of the gut. From these cells six ducts on each side lead forwards, passing dorsal to the oral sucker and opening into the small pit containing the stylet.

The gland-cells and their ducts are filled with small clear granules which stain very deeply with iron haematoxylin, and take up strongly the acid fuchsin in Mallory’s triple stain. The nucleus of each cell is large, with a reticulum of chromatin and a small nucleolus.

—k group of about a dozen large cells, both anterior and lateral to the stylet gland-cells, is situated dorsally in the body. Their nuclei, each containing a round nucleolus, are comparatively small and are poor in chromatin; their dense cytoplasm is packed with numerous minute granules which stain blue with Mallory’s triple stain, and do not retain iron haematoxylin, being always coloured by the counterstain. In these respects they are quite distinct from the stylet glands ; they may well be the true cystogenous cells.

Excretory System (fig. 2, Pl. 11).—On each side of the body a main anterior lateral duct arises from a group of three flame-cells, and leads backwards, being joined by the ducts of three more flame-cells.

Each posterior lateral canal arises similarly from a group of three flame-cells, and leads forwards, receiving the ducts from nine more flame-cells which do not appear to be arranged in definite groups.

There are thus eighteen flame-cells on each side of the body, each cell being of the usual type found in adult Trematodes (Text-fig. 3).

The anterior and posterior lateral canals join on each side at a point a short distance in front of the ventral sucker, and from each junction a much-coiled duct leads backwards to the anterior end of the excretory vesicle. The excretory vesicle is a wide canal enclosed by a layer of large columnar cells which are described below. Amongst the ends of these cells, abutting on the lumen of the vesicle, run muscle-fibres which join to form an irregular network; these fibres contract at intervals to discharge the contents of the vesicle through the posterior, dorsally placed excretory pore. A sphincter of strong circular muscle-fibres closes the narrow canal between the vesicle and its pore.

One of the most conspicuous features of this and other gorgoderine cercariae is that the lumen of the excretory vesicle is lined by a layer of large columnar cells, occupying most of the posterior body ; in life, these are packed with round clear granules.

Stages in the development of these cells were followed in material fixed with ‘Flemming’s without acetic’, sectioned at 4p. and stained with iron haematoxylin counterstained with orange G.

The cells are first recognizable in germ-balls 0·25 mm. long (after fixation), as a closely packed oval mass embedded in the parenchyma posterior to the rudiment of the ventral sucker. The spherical nuclei contain large nucleoli and their chromatin material is in the form of a deeply staining network. The appearance of the nucleus remains constant throughout the further development of the cell. At this stage the cytoplasm is dense, non-granular, and takes up the counterstain strongly.

No lumen is recognizable; in living germ-balls of approximately the same age, there are two main lateral excretory ducts which open separately at the posterior end of the body.

The ducts become parallel for a short distance before they open to the exterior, and in living material they appear, at this part of their course, to pass through the mass of cells which later line the excretory vesicle; since the excretory ducts collapse and become unrecognizable when the germ-ball is fixed, it has not been possible to confirm this on preserved material.

As the germ-ball develops, the parallel portions of the ducts approach one another and fuse to form a single excretory vesicle, lying at the centre of the mass of young ‘lining’ cells, and opening through a single excretory pore. A similar fusion of parallel ducts is described in Cercaria vitrina by Neuhaus (1936).

At this stage the lumen appears in sectioned material, and is surrounded by the cells which now tend to be columnar, the large nucleus being at the outer end of each cell, and the cytoplasm near the nucleus containing large granules. These, however, do not yet stain differentially from the cytoplasm (Text-fig. 5).

Somewhat later, at the time when the cuticle of the young cercaria is being formed, the granules, all still at the nuclear end of the cell, stain strongly with iron haematoxylin. This stage would appear to be of short duration, since in preparations it is found less frequently than the first and third stages (Textfig. 6).

The granules immediately afterwards migrate towards the lumen of the excretory vesicle, and are replaced at the nuclear end of the cell by ‘immature’ granules which do not retain the haematoxylin (Text-fig. 7).

The cells are in this condition at the time when the cercaria escapes from the mollusc; in well-fixed mature cercariae the granules, filling the distal ends of the cells, seem about to be shed into the lumen.

No living cercariae have been observed to discharge granules from the excretory pore, and it may be that the cells are actually cystogenous, as suggested by Ssinitsin (1907) ; but in the few encysted cercariae I have been able to examine the cells were still conspicuous, and still contained many granules ; when the cercaria moved, a few similar granules could be seen floating freely within the cyst. Thus, although positive evidence is lacking, it seems most probable that these cells have an excretory function and that the granules consist of excreted material.

Figs. 1, 2, and 3 are drawn from living material. Figs. 4 and 5 are partly from camera lucida drawings of sections, and partly from living material.

PLATE 11.

Fig. 1.—Mature Cercaria macrocerca with body in its normal position enclosed in proximal part of tail. (× 150.)

Fig. 2.—Ventral view of body showing excretory system. (× 250.)

Fig. 3.—Stylet, viewed from above on right side. (× 2,000.)

PLATE 12.

Fig. 4.—Ventral view of body showing general anatomy. (×250.)

Fig. 5.—Body viewed from right side showing general anatomy. (×250.)

a.l.c., anterior lateral canal ; c.mu., circular muscles ; c.sir., central strand of tail; cut.sp., cuticular spines; d.v.mu., dorso-ventral muscles; e.p., excretory pore; e.v., lumen of excretory vesicle; f.c., flame-cell; g.c., stylet gland-cell; g.c.d., stylet gland-cell duets; gl. 1, mucus cells of tail; gl. 2, group of glandular cells; i., intestinal branch; L.c., Laurer’s canal; I.mu., longitudinal muscle-band; m.e.c., main excretory canal; mu.c., nuclei of muscular cells ; n.g., supra-oesophageal ganglion ; oes., oesophagus ; op.ch., opening of caudal chamber enclosing body; ov., ovary; p.l.c., posterior lateral canal; pr.t., proximal part of tail forming the chamber; s.e.v., sphincter of excretory vesicle; s.p., cuticular sensory papillae; st., stalk connecting body with tail; s.v., seminal vesicle; T.l, anterior testis ; T. 2, posterior testis ; t.mu., muscles radiating from proximal end of tail to walls of body; ut., uterus; vit., vitellaria.