Studies on Tapeworm Physiology: I. The Cultivation of Schistocephalus Solidus In Vitro

The lack of knowledge concerning the metabolism and general physiology of cestodes is mainly due to technical difficulties presented by their in vitro cultivation. Up to the present, attempts to cultivate parasitic stages of helminths have been relatively unsuccessful and in cestodes it is doubtful whether normal development has ever been obtained.

One of the major difficulties has been the absence of accurate information regarding the nutritional requirements of tapeworms, and thus ‘any attempts to feed cestodes in vitro cannot be otherwise than empirical’ (Wardle, 1934). A great variety of media have been used by various workers, including physiological salines—either alone or with the addition of carbohydrates, nutritive broths, serum, tissue culture media, etc.

Bacterial contamination is, however, the most important controlling factor in this type of cultivation. Since adult cestodes with one exception (Archigetes sieboldi, parasitic in the body cavity of oligochaetes) are all parasites of the alimentary canal of vertebrates, this difficulty is one of paramount importance, as the mucus film surrounding the worms is invariably rich in microflora. Rinsing the cestodes in dilute solutions of various bactericidal substances have met with little success (Wardle, 1934). The sedimentation technique—used with success by Ferguson (1940) for obtaining sterile trematode cercariae—has produced better results, and Wardle & Green (1941) succeeded in maintaining undulant activity in Hymenolepis fraterna without bacterial clouding for 20 days in dilute Baker’s medium. In general, however, experiments with adult cestodes have been most unsatisfactory.

Larval cestodes being frequently localized in aseptic host tissues have given much more promising results. It seems likely that if a technique for the aseptic cultivation of larval tapeworms in vitro could be devised, and the conditions for the growth and development of the larva to the strobilar stage established, the problem of obtaining adult cestodes in a sterile condition would be largely solved.

The pseudophyllidean cestode Schistocephalus solidus Müller has many advantages for experiments of this nature, since its plerocercoid larva occurs in the body cavity of the three-spined stickleback Gasterosteus aculeatus, where it lives under completely aseptic conditions.

The problems to be solved briefly are : (a) the development of a technique for the removal of the larvae from the body cavity of the fish without bacterial contamination; (b) the provision of a suitable medium allowing the larvae to survive long enough for physiological experiments to be carried out; (c) the production of an appropriate stimulus to convert the larvae into adult forms.

Frisch (1734) kept the plerocercoid larvae of Schistocephalus alive for 2 days in river water, and Abildgaard (1793) stated that the same larvae could be kept alive for 8 days in fresh tap water.

With regard to other cestodes, both larval and adult, a number of references are available, but only the more successful will be mentioned here. The results of culture attempts are summarized in Table 1. For a more detailed account of some of the earlier work, reference may be made to the reviews of Wardle (1937 b) and Hoeppli, Feng & Chu (1938). Among the earlier workers, the most successful results were those of Lônnberg (1892) who kept Triaenophorus tricuspidatus alive in a dilute pepsin-peptone solution for over a month. More recently, Dévé (1926) kept the larvae of Echinococcus granulosus from the lung and liver of sheep alive for 2 weeks in aseptic hydatid fluid plus untreated horse serum. Coutelen (1927), using the same larvae and a medium of hydatid fluid plus various nutritive media, reported a viability of 31 days, and later (1929) kept Coenurus serialis, from rabbits; alive for 20 days, using normal saline plus fresh serum. The results of both Dévé and Coutelen are of particular interest as a transformation of the scolices of the cysticercoids was obtained, with considerable increase in size and bladder formation. Stunkard (1932) observed an increase of 3–4 times in length in larval Crepidobothrium lonnbergi kept alive for 32 days in dextrose-saline plus broth; segmentation of the strobila also took place, but only sterile and abnormal proglottids developed. The longest recorded viability in vitro for any cestode—larval or adult—was that obtained by Mendelsohn (1935) who cultured the larvae of Taenia taeniaeformis from a rat’s liver for 35 days in sterile saline plus chicken embryo extract and filtered horse serum.

Wardle (1932, 1937a), working with the plerocercoids of Nybelinia surmenicola, Triaenophorus tricuspidatus and Diphyllobothrium latum under oligoseptic conditions using sterilized media, showed that, although complete asepsis was never obtained, the viability with unchanged sterile salines was considerably higher than with unsterilized media changed frequently. Larval Nybelinia gave the best results as they remained viable in double-strength Locke’s solution up to 19 days. Earlier results with solid media were not satisfactory (1934), but recently Wardle & Green (1941) have reported the cultivation of the plerocercoids of Diphyllobothrium latum on various nutrient agar media, and in one experiment, using agar plus hog serum, considerable growth was obtained, although the total viability was only 5 days.

Joyeux & Baer^* (1942) have investigated the cultivation of the plerocercoids of Ligula, a cestode closely allied to Schistocephalus. Larvae were cultured in Petri dishes at 38–40° C. in (a) saline, (b) saline plus ascitic fluid, (c) saline plus horse serum, (d) saline plus serum and ascitic fluid. In the last three media, larvae remained viable for a maximum period of 12 days, and seven out of the twenty-four larvae used became apparently sexually mature and underwent oviposition. Sections revealed that normal development had not taken place, since spermatogenesis had not occurred. The addition of a number of tissue extracts were tried out in an attempt to induce spermatogenesis, but with negative results.

Wilmoth (1945) used a very wide range of simple and complex media in an attempt to find a suitable culture medium for the cysticercus of Taenia taeniaeformis, but with little success. Survival of this larva was longer in simple than complex media, and maximum viability of 24 days was obtained in Ringer’s solution plus a trace of glucose.

According to Joyeux & Baer (1936), the strobilar phase of Schistocephalus occurs most commonly in mergansers and domestic ducks, but it has also been reported from numerous other birds frequenting marine and fresh-water areas. The adult is remarkable in that it only spends a very short period of its life cycle in the gut of the bird host, with the result that this phase is rarely found.

Kiessling (1882) fed a number of infected sticklebacks to domestic ducks and found that all the contained Schistocephalus were passed out with the faeces within 3-4 days. Unlike most other cestodes, the posterior proglottids are not cast off when ripe, but the entire worm is shed. The eggs are ejected from the uterine pores and pass out with the droppings of the bird.

The organism has the usual pseudophyllidean type of life cycle. The eggs which reach water hatch out in about 3 weeks into typical ciliated hexacanth embryos of the bothriocephalid type (Schauinsland, 1885). The free-swimming embryos are eaten by Cyclops spp., in the body cavity of which they develop into procercoids—as many as 60 having been found in a single crustacean. Nybelin (1919) experimentally infected C. serrulatus and C. bicuspidatus, and Callot & Desportes (1934) likewise infected C. viridis.

When infected Cyclops are eaten by a stickleback, the contained procercoids migrate through the intestinal wall of the latter and enter the body cavity where they develop into plerocercoids. Although the stickleback is the normal host for the plerocercoid phase, it has also been reported from Atherina mochon and Blennius vulgaris (Forti, 1932). Birds become infected by eating fish containing plerocercoids.

The fish used in the present experiments were collected with a hand net from a large pond at Hunslet, Yorkshire. Throughout the course of this work several hundreds of fish were examined. Every fish collected from this area was found to be infected although the number of larvae per fish varied greatly. 4–10 larvae was a common number recorded, although a heavy infection of up to 140 was occasionally found.

Even the smallest fish collected (about 2 cm. in length) invariably contained one or more of the larvae. Infected fish show a very characteristic swelling of the abdomen which produces unnatural swimming movements. The plerocercoids lie in the body cavity (Pl. 2, fig. 1) packed close along the sides of the gut and between the other viscera which invariably are much compressed. Considering the extent of the infection—in some cases up to 40% of the entire weight of the fish—it is surprising that the larvae do not have a more apparent effect on the fish which reach their normal size in spite of the parasites. The plerocercoids of the closely allied form Ligula intestinalis produce temporary castration in Atherina mochon, but there is no evidence that the same condition occurs with Schistocephalus.

Infected fish were kept in the laboratory in fresh-running water under suitable conditions (Craig-Bennet, 1930) and killed as required by pithing. The technique for obtaining sterile larvae from the fish is somewhat elaborate, and some practice was necessary before aseptic cultures were obtained. The pithing was carried out by a long fine needle. The latter was pushed carefully and firmly down the neural canal so that a secure hold could be obtained on the fish for further manipulation without touching the skin. During the pithing, the fish was held by the skull only, as in heavy infections the larvae were forced out prematurely by the slightest pressure on the abdomen, rupturing the rectum and emerging through the anus with consequent faecal contamination.

Although the larvae lie in aseptic surroundings in the body cavity, it is further essential to sterilize the surface of the skin which is rich in microflora and with which the larvae may come in contact during the process of extraction. The skin was carefully dried with a soft cloth, and its entire surface painted with two coats of a saturated solution of iodine in absolute alcohol. The handle of the needle embedded in the fish was next secured in a bench vice, so that the fish was in a horizontal position with the ventral side towards the observer. The hands of the latter were thus freed for dissection. The upper part of the body cavity was slit open carefully with a sterilized cornea knife and the larvae, pressing closely against the body wall, immediately emerged to the outside; they were manipulated into the mouth of the tube containing the medium under investigation by means of a flamed platinum loop. It is important only to remove larvae from the anterior part of the body cavity, as worms in the posterior region may easily become infected by faecal droppings which sometimes exude through the anus. Care must also be taken to avoid puncturing the alimentary canal with the platinum loop during the manipulation.

The cultivation was carried out in plugged 15×1·5 cm. test-tubes kept away from direct light at laboratory temperatures. Petri dishes were found to be unsuitable for cultivation as they became easily infected. As Wardle (1934) found Locke’s solution (NaCl 9·0 g. ; KC1 0·42 g. ; CaCl 0·24 g. ; NaHCO₃ 0·2 g. ; distilled water 1000 c.c.) the most satisfactory saline medium for plerocercoids, various dilutions of this medium—hereafter referred to as Locke—were used both pure and with the addition of glucose. The dilution is indicated by a prefix; thus $32$ Locke represents $112$ times normal Locke’s solution. The complete list of media used is shown in Table 2. As a nutrient medium, peptone broth was chosen ; it was prepared from ox hearts following the usual procedure for bacteriological cultures (Bigger, 1935).

The medium in each tube was changed every 30 days, the renewal being carried out by means of a sterilized pipette. Owing to the refractive effects of the tube curvature, larvae could not be observed directly with a dissecting microscope. By partly dismantling and rearranging the microscope, however, it was possible to over-come this difficulty by viewing the reflection of the larvae from below (Text-fig. 1).

During the first day of cultivation, the larvae were examined for viability every hour; subsequently they were examined every 24 hr. Plerocercoids which were viable showed very marked undulant activity when subjected to a strong light for a few minutes—possibly a result of radiant heat effects rather than light. If no undulation was observed, the viability was further tested by warming the tube by holding it in the vicinity of a small Bunsen flame for a few seconds; if alive, the larvae immediately responded to the stimulus. Larvae which failed to respond to this latter stimulus were considered dead.

The percentage of aseptic cultures obtained by this method varied from 75 to 100%. Cultures accidentally infected were easily recognizable as they became foetid and clouded within 2-6 days. In order to ascertain whether any histological or anatomical change had taken place during cultivation in the various media, larvae were fixed at intervals. Bouin was used for routine histological work and Carnoy and Champy for more detailed cytological study. Sections were stained in Heidenhain’s iron-alum haematoxylin or Delafield’s haematoxylin and eosin, the former giving much superior results.

This preliminary series of experiments aimed at establishing a satisfactory aseptic technique as well as giving some indication of a suitable culture medium. When this was completed, a second series of experiments was carried out using the more favourable media incubated at the mean body temperature of the duck, 40° C. (Wetmore, 1921), in an endeavour to induce further development as in the bird host.

Schistocephalus solidus Miiller, 1776 is the only species of the genus Schistocephalus Creplin, 1829, belonging to the subfamily Ligulinae Lühe, 1879, of the family Diphyllobothriidae Lühe, 1910. This species is unique among cestodes in that the plerocercoid phase is completely segmented.

The anatomy of the plerocercoid was studied from whole-mount preparations and from reconstructions from serial sections. Except for minor points, it agrees in general with that described by several authors (Moniez, 1881; Kiessling, 1882; Linton, 1927).

A typical well-developed plerocercoid (Pl. 1, fig. 1) consists of an anterior bothrial segment (b) followed by some 60-80 proglottids. In the larva as removed from the fish, the bothrial segment is bluntly rounded with a small anterior pit (Text-fig. 2A). During artificial cultivation this bothrial segment becomes everted and assumes the typical sharply-pointed adult condition (Text-fig. 2B). This eversion usually also occurs in whole-mount preparations (Pl. 1, fig. 1) on account of compression during fixation which is carried out between glass plates. The size of the larvae varied from 0·1 to 5·0 cm. (extended length)—the most frequently occurring length being between 2 and 3 cm. The genitalia reach a high grade of development in the larvae. A few clusters of testes cells appear in the 9th proglottid and the rudiments of the cirris can be seen in the nth proglottid. From the 12th proglottid backwards, in forms larger than about 2 cm., the remainder of the genitalia are present in every segment. In some larvae, the genitalia in the most posterior three or four segments (Pl. 1, fig. 1, a.) are imperfectly developed, and although the rudiments of the cirris and the uterus are present, testes and yolk glands are either absent altogether or represented by a few cells only.

Histological examination reveals that the apparent advanced development of the genitalia is in fact superficial, with the cells of the reproductive organs in an immature condition. The ovaries (Pl. 2, fig. 4, 0. ; Text-fig. 3), lying in the lateral fields of each proglottid, consist of a compact mass of oval cells with large nuclei and little cytoplasm. A few cells are sometimes seen undergoing mitosis, but the number of actively dividing cells is small ; meiosis has never been observed in the larval ovary. The oviduct is fully developed. The yolk glands are seen in sections as small unconnected clusters of round or spindle-shaped cells spread around the periphery of each proglottid except in the region of the genital pores (Pl. 2, figs. 2, 4, yg.). Each cell contains a large nucleus with a heavily staining nucleolus (Pl. 3, fig. 1 ; Text-fig. 5A); the cytoplasm is lightly staining and free from the typical granular appearance associated with yolk formation.

The testes which lie in the parenchyma on the side opposite the genital pores are enclosed in thin-walled membranes. Their condition varies somewhat in different larvae. For the most part they consist of a mass of oval or spindle-shaped cells which sometimes fill the entire capsule (Pl. 3, fig. 4) but which show no signs of differentiation. The number of cells, however, varies quite considerably, and in some testes there is a well-defined cavity. Mitosis is comparatively rare but has been seen in some specimens. The condition of the larval testes is therefore very primitive, and although the variable number of cells indicates that the cells can increase in number, no spermatogenesis takes place.

The cirris and seminal vesicle are fully developed, but the vas deferens, contrary to the description of Kiessling (1882), is single and shows no connexion with the testes at this stage but tails off into the parenchyma (Text-fig. 2C; Pl. 2, fig. 2). Even in the best preparations under oil immersion, no vasa efferentia could be located.

Since both medium-sized larvae of about 60 segments (approximately 2 cm.) and very large larvae of 80 segments (approximately 5 cm.) show genitalia in the condition described above, it is evident that the larvae can grow in size and increase the number of proglottids during their stay in the fish, but seemingly the genitalia cannot undergo any degree of differentiation or maturation until taken into the gut of the final bird host. As will be shown later, providing the food requirements are fulfilled, the stimulus for maturation of the genitalia is provided by the change in temperature between the fish and bird hosts.

The number of larvae surviving at intervals during cultivation at room temperatures in the various media are shown in Table 2. It must be emphasized, however, that the important factor to be measured is the length of time the larvae behave normally rather than the total viability in vitro. With the exception of the very toxic hypertonic media, two main abnormal features appeared after prolonged cultivation in all the media: cuticular peeling, and degeneration.

Cuticular peeling commenced by a loosening of the outer layer of the cuticle (Pl. 1, fig. 3) at the posterior end of the organism and in extreme cases spread over the entire surface of the worm until the cuticle was completely stripped off. The extent of the cuticular peeling varied greatly, and in the most favourable media was barely noticeable and appeared only after very prolonged cultivation.

Degeneration commenced at the posterior end and gradually extended forwards. The tissue of the abothrial proglottids became flaccid and sometimes even transparent, although at the same time the anterior part of the organism remained active and exhibited marked undulation for some considerable time.

The appearance of these two features was used as a criterion forjudging the period of normal behaviour. It is difficult, however, to decide the exact moment of transformation from normal to abnormal behaviour, so that the figures for the duration of normal behaviour in vitro given in Table 3 can only be considered accurate to within 2 or 3 days.

Nutrient peptone broth gave the most satisfactory results of all the media used, in that it gave the highest period of viability and normal behaviour, although the percentage of larvae surviving more than 140 days was less than in some of the salines. The activity in this medium was very marked, and cuticular peeling and degeneration, when they did occur, were slight. Of the twenty larvae used, seven were normal and active after 140 days’ cultivation and one is still alive and exhibiting normal behaviour after 300 days’ cultivation.

Of the various saline media tested, $34$ Locke gave the most satisfactory results with a mean period of normal behaviour of 114 days; nine out of the ten larvae used surviving for more than 140 days.

It is of interest to compare the behaviour of the larvae in the series of salines tested :

All larvae in 2 Locke, $32$ Locke and $54$ Locke died within 4 hr. During the first few minutes of immersion they showed only slight activity, followed rapidly by tetanic contraction accompanied by a twisting and contortion of the organism and very marked cuticular peeling. There was no degeneration.

In normal Locke, larvae showed some contraction on first immersion, but regained their normal appearance after several hours. Cuticular peeling commenced in some larvae after 24 hr. but only to a very slight degree. The majority of larvae remained active though somewhat sluggish for 60 days; seven larvae survived for more than 140 days, but the maximum period of normal behaviour was 114 days. The addition of 1 % glucose to the medium gave a higher maximum period of normal behaviour, but the mean was lower. The behaviour in normal Locke plus 0·5 or 0·25% glucose was very unsatisfactory, all the larvae dying within 50 days.

In $34$ Locke, the larvae behaved in a very normal manner, and even after 100 days’ cultivation all the organisms reacted violently to light and in fact appeared little different from larvae freshly removed from the fish. Very marked degeneration, however, began after 106–131 days.

In $34$ Locke + 1 % glucose, there was normal behaviour for the first 22 days, after which the majority of larvae soon became sluggish and all died within 70 days. In $34$ Locke + 0·5 or 0·25 % glucose, the activity in the early stages was not so marked as in pure $34$ Locke, and cuticular peeling in general was apparent after only 8–13 days, though a small proportion of larvae remained viable and normal for over 100 days.

Some 30% of the larvae lived for over 140 days in $12$ Locke, but the activity was not so marked and cuticular peeling commenced in some cases within 7 days. The hypotonicity of the medium produced considerable relaxation of the musculature, and the larvae became extended to about twice the length in the fresh condition. Behaviour in $12$ Locked-1 or 0·5% glucose was unsatisfactory, as about 50% of the plerocercoids died after 10 days ; only one of the sixty-three used survived for 23 days. The addition of 0·25 % glucose gave somewhat better results, and although some larvae died within 8 days, four survived for more than 90 days and one for over 140 days. The general activity throughout, however, was sluggish, with the result that the normal behaviour time was low.

An interpretation of these results is difficult in view of the inconsistencies in the viability of the larvae in any one medium. For example, nineteen out of the twenty-eight larvae cultured in $34$ Locke+ 0·25% glucose died after 20 days’ cultivation, yet four lived for over 100 days, and one for over 140 days. It is probable that there are marked inherent differences between individual larvae or that there are many as yet unknown factors playing a part in the control of viability. The range of viability does, however, compare with that obtained by Brand & Simpson (1942) in cultivation attempts with nematodes.

The osmotic relationships of the organism to the medium is a matter of some importance, and a subsidiary series of experiments was therefore carried out in order to obtain some knowledge on this point. The determination of the exact osmotic pressure of helminths in general presents a difficult problem. Schopfer (1932) prepared ‘tissue extracts’ of various cestodes and nematodes and with elaborate apparatus estimated the osmotic pressure cryoscopically. Estimation of volume change is a simple method easily applied to forms such as plerocercoids. The method involves the measurement of the change in weight after immersion in saline solutions of varying osmotic pressure. The main difficulty is to obtain ‘reproducible removal of adhering water’ (Krogh, 1939), but with care an accuracy of 2-3 % can be obtained with this method.

After careful drying on filter paper, a number of larvae were weighed and immersed in solutions of 0·5, 0·6, 0·7, 0·75, 0·8, 0·9 and 10% ‘Analar’ sodium chloride in specially cleaned Petri dishes. They were removed, dried and weighed at intervals of 30 min. The results from two series of experiments are given in Table 4 and expressed graphically in Text-fig. 4.

It is only possible to base estimations on short periods of immersion, as the behaviour becomes abnormal after 2-3 hr. in the higher and lower saline concentrations as already noted. It may be assumed that the change in volume is proportional to the change in weight, as density and metabolic changes are small enough to be neglected.

From the curves it is evident that the zero change in weight occurs in concentrations of sodium chloride approximately between 0·7 and 0·8%. The actual readings from the curves were: 0·72 (90 min.), 0·75 (60 min.), 0·78 (30 min.), with a mean of 0·75 ± 0·3 %. It is convenient to express the osmotic pressure of solutions in terms of the depression of the freezing-point (Δ). Taking into consideration the correction factor for the dissociation of sodium chloride at this concentration (International CriticalTables, 4, 1929), the value for the osmotic pressure of Schistocephalus is Δ = −0·44 ±0·02° C.

Histological examination of the larvae cultured in any of the media for over 100 days at room temperature revealed that no further development had taken place, the condition of the genitalia being precisely the same as that of fresh larvae as removed from the fish. Throughout the entire cultivation period, no evidence of growth was recorded. The worms were measured as accurately as was possible through the walls of the culture tube and again after prolonged cultivation; the marked undulation made exact measurement difficult. It was not possible to weigh the larvae, owing to the technical limitations imposed by the maintenance of aseptic conditions. Nor was it practicable to count the number of proglottids of larvae within the tubes. It is possible, of course, that the degree of growth was so small as not to be measurable within the limits of the somewhat large experimental error involved, but further investigation is needed to decide this point.

The object of cultivating the larvae in vitro at the body temperature of the final host was to determine whether the sudden increase in temperature alone would provide sufficient stimulus to induce further development and maturation of the genitalia.

Within 30 min. of the beginning of incubation in broth in tubes placed in an oven at 40° C., larvae became extremely active and expanded and contracted with such vigour within the tubes that the top of the medium often became frothy and the tubes even vibrated slightly. The bothrial extremity became completely everted and pointed (Text-fig. 2B) as in the adult form. Cuticular peeling commenced within an hour, and after about 24 hr. cultivation the entire cuticle was shed from the worm. Apart from these features, no noticeable external feature was observed until about 48 hr. later, when eggs began to appear at the uterine pores in the anterior segments containing genitalia. Within a few hours, with the exception of the first eleven segments, every proglottid was ejecting eggs at a rapid rate. The oviposition continued for 2–4 days, the activity gradually diminishing. At the cessation of oviposition the exhausted worms died—the total period of viability in vitro under these conditions being 4–6 days.

During oviposition the eggs tended to accumulate in little clusters around the uterine pores, plainly visible as lightish brown patches in the centre of each pro-glottid containing genitalia. By gentle shaking the eggs were freed and allowed to settle to the bottom of the tubes.

At higher magnification it was further possible to observe not only ejection of the eggs but also the actual sexual process of the cirris—unseen under low power, as the cirris is a very delicate minute structure almost invisible against the white background of the body of the worm. The vigorous undulation of the organism in the medium makes this observation even more difficult, and considerable practice manipulating the light was necessary before it was satisfactorily observed in detail.

Eversion of the cirris commenced at 40–60 hr. after the commencement of incubation. The cirris region became swollen and formed a slight elevation in each proglottid. This swelling later began to ‘throb’ in a regular manner and finally the cirris everted completely. Once the eversion process started it continued for about 30 hr. During the sexual process, the cirris everted directly outwards; there was no attempt at a sideways movement towards the vaginal pore. The cirris everted and invaginated at the rate of about once per second.

The actual discharge of spermatozoa was not observed, but it can be assumed that they were ejected directly into the culture medium at each eversion, as microscopic examination of the debris at the bottom of the tube revealed their presence. Later experiments with two or three larvae within a culture tube, making cross-fertilization a possibility, gave precisely identical results. Although the worms were frequently in quite close apposition due to confinement within the tube, the cirris always everted directly outwards from the proglottids and in no instance did copulation occur between the worms.

Histological examination revealed striking changes in the internal organization with complete maturation of all the organs previously in a primitive immature condition. Worms were fixed at intervals of 24, 48 and 72 hr. cultivation. As the present problem is concerned mainly with the physiological aspects of the development, detailed cytological examination was not undertaken in this investigation.

• (a) Female genitalia. The ovaries show the least degree of change of any of the genitalia. The nuclei, which in the larval stage are either resting or undergoing slow mitotic division, now show meiotic figures especially in the region of the junction with the oviduct. The uterus—narrow and thick-walled in the plerocercoid—becomes greatly enlarged and distended with masses of eggs and free yolk cells (Pl. 2, fig. 5, ut.). The yolk glands undergo marked changes. After 24 hr. incubation, the cytoplasm becomes filled with small basiphil granules often accumulated at one end of the cell (Text-fig. 5B). After 48 hr. the cells have become fully developed (Text-fig. 5 C) and have increased in size from 9 × 4μ to 13 × 10μ (mean values). Numerous globules of yolk of varying sizes and degrees of staining are present in the cytoplasm. The swelling of the yolk cells causes the glands to lie in close apposition so as to appear fused together and form a continuous band around the periphery of each proglottid except in the immediate region of the genital pores (Pl. 2, figs. 3, 5). After Carnoy fixation the fatty component of the yolk becomes dissolved out, so that the granules seen in Text-fig. 5C represent only the protein base of the yolk. In fresh yolk cells, seen in teased-out tissues, the yolk globules appear as yellow refractive spheres which completely fill the cytoplasm. In Champy-fixed material they appear as brownish black globules. No attempt has been made to follow out the origin of the yolk during its formation. One fact, however, is certain, the yolk is formed from the cytoplasmic inclusions and not by nucleolar extrusion, as the nucleolus remains apparently unchanged during vitellogenesis. The eggs as laid varied in size between the limits—lesser diameter 35–46μ., greater diameter 58–75 μ, with a mean value for ten eggs of 38 × 64μ. These values agree closely with those of Linton (1927), who from preserved specimens obtained 36–42 μ, and 57–69 μ for the lesser and greater diameters respectively.

• (b) Male genitalia. Sections of the testes revealed that complete spermatogenesis had taken place within 40-50 hr. incubation. Every testis capsule contained numbers of mature spermatozoa together with spermatocytes and spermatids in progressive stages of maturation. Spermatogenesis appears normal in every way and follows the typical pattern. A sperm-mother cell by repeated mitotic division gives rise to an 8-celled morula. Meiosis takes place at this stage and results in a 32-celled spermatid morula. The centriole in each spermatid which was not previously visible now appears as a deeply staining granule on the peripheral side of each cell (Pl. 3, fig. 3, sr.) at the same time as the nuclei begin to elongate. By further elongation (Pl. 3, fig. 3, sd.) the nuclei develop into the typical narrow sperm heads, and the tail arises by outgrowth from the centriole and grows rapidly back. The minute size of the male germ cells makes observations on the chromosomes difficult, but it is tentatively suggested that the haploid number lies between 6 and 8 and the diploid between 12 and 16; these numbers, however, are subject to confirmation.

The mature spermatozoa lie in typical whorls in the lumen of the testis capsules. They have little affinity for nuclear stains and can only satisfactorily be seen in Carnoy-fixed material heavily over-stained in Heidenhain’s haematoxylin (Pl. 1, fig. 2). The vasa efferentia, which are apparently undeveloped in the plerocercoid, are easily visible in the mature worm as very much coiled tubes filled with spermatozoa. The seminal vesicle, which is small in the larva, becomes greatly enlarged and is packed tightly with spermatozoa.

In addition to the normal stages of spermatogenesis described above, the majority of testes contain a very large cell with polypoid chromatin material. These giant cells measure between 10 and 15μ in diameter and contain a very large number of chromosomes—so large, in fact, that it has not been possible to count them in existing material. These cells invariably lie free in the lumen of the testes. There is no evidence to suggest what role they play in the process of spermatogenesis. They may act as ‘nurse’ cells of some kind, or alternatively they may simply be abnormal cells produced by the artificial conditions of cultivation. Unfortunately, specimens of normally matured adult Schistocephalus from the bird host were not available, so that comparison between the normal spermatogenesis and that induced by artificial cultivation was not possible.

In contrast with the results obtained in peptone broth, the incubation of plerocercoids in salines or glucose salines did not induce the fully mature condition. In the great majority of cases worms died within 1–3 days after a period of intense activity. Only in one out of the ten larvae used did spermatogenesis and partial vitellogenesis take place. In no case did the cirris undergo eversion, although in the larva in which spermatogenesis had taken place, fully formed spermatozoa were found in the testes. Apart from the partial granulation of the yolk glands in this same larva, indicating that vitellogenesis had commenced, the female genitalia showed no appreciable change. The ovaries showed no meiosis and oviposition did not occur.

After oviposition was completed the mature worms were removed from the media and the tubes gently shaken to stir up the accumulated eggs at the bottom. The broth, together with the eggs, was transferred to a sterile centrifuge tube and centrifuged. The eggs were washed with several changes of sterile water and divided into two portions. One portion was incubated in water in sterile centrifuge tubes in an oven at 25° C. ; the other was placed in a few drops of water in a watch-glass in a covered Petri dish exposed to sunlight, after the method recommended by Rosen (1920).

No further development of the eggs was obtained in either case, even after 6 weeks’ cultivation. The normal time of development at room temperatures is about 3 weeks (Callot & Desportes, 1934), but in Ligula the eggs hatch out in 8 days in the warm summer months (Joyeux & Baer, 1942). Microscopic examination of the eggs revealed that they had undergone no internal development, although the enormous amount of yolk in the eggs of Schistocephalus makes observation of the internal changes difficult.

In view of the fact that the worms had not apparently copulated during cultivation, it was at once suspected that the failure of the eggs to hatch was due to non-fertilization. A careful re-examination of sections of the sexually mature worms showed the absence of spermatozoa from the vagina ; nor were any stages that could be interpreted as representing fertilization found. It must be emphasized, however, that on account of their minute size and poor staining affinity, identification of spermatozoa—except in large masses—is one of some difficulty. That fertilization had not taken place was further confirmed, however, by the fact that the ova as seen in sections of the uterus contained apparently the haploid number of chromosomes only.

In assessing the suitability of a medium for the artificial cultivation of cestodes in vitro, four main criteria must be taken into consideration—viability, normality of behaviour, growth and development.

With the exception of growth, for which there is no definite evidence, peptone broth answers all the other criteria for suitability as a culture medium for both the plerocercoid and the strobilar phase. To some extent, too, even such a relatively simple non-nutrient solution as $34$ Locke allows of considerable viability and normality of behaviour, and would be a suitable medium for studying the physiology of the plerocercoid stage, but not the strobilar.

In these empirical investigations, with the exception of the osmotic pressure, physical factors, such as oxygen tension, pH, and surface tension, have not been taken into consideration, and further investigation will be required to determine whether they play a significant role in the control of viability. The high viability obtained suggests that, like nematodes, cestodes may be relatively insensitive to a wide variation in environmental factors (Brand & Simpson, 1944). Moreover, throughout the experiments, the media in each tube was only changed every 30 days, and it may be that if the medium was renewed more frequently, considerably higher periods of viability of the plerocercoids could be obtained, as it seems not improbable that the by-products of metabolism may have some cumulative toxic effect.

The viability of the mature Schistocephalus (4–6 days) in peptone broth at 4O°C. is especially significant, as this period, although apparently brief, is equal to the normal longevity of the adult in the natural bird host; so that, in fact, it is possible to culture the adult Schistocephalus in vitro for a period equivalent to its viability in vivo.

The fact that plerocercoids can live and behave normally in pure saline media containing no nutrient substance points to a very low metabolic rate in the larval condition. This is not surprising, in view of the fact that for the most part larvae remain motionless within the culture tubes until stimulated by light or heat. Furthermore, since they are undergoing no reproductive processes, the total expenditure of energy will be small. During all this period it is evident that larvae must be drawing on their reserve food supplies. It is well known that the food reserves of cestodes are mainly in the form of glycogen, representing some 20–30% of the dried weight (Brand, 1933 ; Smorodinzew & Bebeschin, 1935 ; Wardle, 1937a; Reid, 1942). This glycogen is stored in the intercellular spaces in the parenchyma (Ortner-Schonbach, 1913; Wardle, 1937a). Many workers believe that glucose is the main carbohydrate requirement of cestodes, and that glycogen reserves are built up from the absorbed glucose. Wardle (1937a) determined chemically the glycogen content of isolated proglottids of Moniezia expansa before and after immersion in saline solutions containing polysaccharides. The most significant result was obtained with Tyrode solution plus glucose, in which proglottids immersed for 6 hr. showed a glycogen gain of 25 mg./g. of fresh weight. Wardle suggested that the glycogen served as a ‘source of fuel for the longitudinal musculature when the latter is maintaining the condition of muscular tonus which characterizes the normal worm’.

In the light of these results it is interesting to note that the viability and period of normal behaviour of the plerocercoids of Schistocephalus was considerably lower in glucose salines than in the corresponding pure salines, with the exception of one worm in normal Locke plus 1 % glucose. However, for the most part the range of viability of the larvae in glucose salines and pure salines was so very considerable that it is not possible to treat the figures statistically to obtain a quantitative result for the addition of glucose. It is advisable, therefore, at this stage not to draw any very definite conclusion from these experiments with glucose salines, as there are undoubtedly many other viability factors as yet unconsidered, and only when all these factors are known and carefully controlled will a quantitative determination be possible. It is certain, too, that there will be some natural degree of variation between individual larvae, but as already pointed out, the results in general are comparable with the range of variation of the viability of parasitic nematodes during cultivation in vitro (Brand & Simpson, 1942).

The behaviour of larval Schistocephalus in salines is in general agreement with the findings of Wardle (1934) for larval cestodes, and of Child and his school (1924, 1926) for free-living platyhelminths. The process of degeneration after prolonged cultivation is similar to that described by Wardle for the plerocercoids of Diphyllobothrium in oligoseptic sublethal media. Degeneration commences at the abothrial segment and extends forwards. According to Wardle, this is due to the fact that the region of maximum activity (in this case the bothrial end) is inhibited first by the penetration of the ions H′, K′ and Na′, which induce tonic muscular contraction, and disintegration then commences at the region farthest away from the inhibited region towards which it gradually extends.

The very high degree of natural behaviour and viability shown in $34$ Locke with an osmotic pressure (Δ=–0·42° C.) approaching that of Schistocephalus as determined by experiment (Δ=–0·44 ±0·02° C.) suggests that the osmotic pressure is an important factor to be considered in the choice of a culture medium. The osmotic pressure of fresh-water teleosts is in the region of Δ =–0·54° C. (Scott, 1916), and it might be expected that media with an osmotic pressure approaching this figure might more nearly reproduce the conditions in the coelomic cavity of the fish. With salines, at least, this is not the case, as activity was much more marked in $34$ Locke than in normal Locke (Δ=–0·56° C.). In peptone broth (Δ=–0·56° C.), however, the duration of natural behaviour was considerably longer than in $34$ Locke, but in this case nutritional factors are also concerned, and it is not justifiable to compare the media on the basis of osmotic relationship alone. It is interesting to note that the osmotic pressure of bird tissues (Δ =–0·56° C., Scott 1916) is close to that of fresh-water teleosts, and thus when the plerocercoids of Schistocephalus are taken into the alimentary canal of the bird host, the change in the osmotic pressure of the environment will be slight. It is possible that a study of the osmotic pressures of cestode larvae and that of their hosts might throw some light on the problem of host specificity, although this does not exclude other factors.

It is difficult to decide whether growth of the larva is actually taking place during the cultivation at room temperature, owing to the limitations in the means of measurement employed. Sections revealed that although the cells of the testes and ovaries sometimes show mitosis, no actively dividing somatic cells were found throughout the entire length of the larvae examined. The only reliable criteria of growth are either increase in weight or number of proglottids, and further technical difficulties will have to be overcome before it will be possible to measure these with the required degree of accuracy, and the question of growth finally decided.

The result of incubating the plerocercoids of Schistocephalus at 40° C. raises a number of interesting questions. Since full development resulting in oviposition took place only in nutrient broth and not in saline or glucose-saline media, it can be inferred that although the sudden rise in temperature undoubtedly provides the stimulus for further development, complete maturation can only be reached providing sufficient nutriment is available in the medium. This is not surprising, in view of the fact that the eggs of the pseudophyllidean cestodes are very rich in food reserves in the form of yolk. The latter provide nourishment for the ciliated embryo during its free-swimming existence. The food requirements of the worm during incubation at 40° C. must therefore be of a high order, as in addition to the production of yolk, fuel must be provided to maintain the great muscular activity which takes place at this high temperature. In pure salines the helminth must rely on its own natural food reserves which evidently are not quite sufficient to fulfil the requirements at this high metabolic rate. On this account it is difficult to account for the fact that one specimen of Schistocephalus underwent complete spermatogenesis and partial vitellogenesis in pure $34$ Locke, whereas the remainder showed no development. It is possible, of course, that this specimen may have carried away an excessive amount of coelomic fluid when it was being removed from the fish. It is remarkable that Joyeux & Baer (1942) failed to obtain spermatogenesis in their experiments with Ligula in vitro at 38–40° C., although a wide range of nutrient media were employed.

There seems little doubt that the infertility of the eggs is due to the failure of normal copulation to take place. Under artificial conditions of cultivation, the spermatozoa are apparently ejected directly into the culture media and fail to make their way to the vaginal pore. It is possible that there may be some sucking action on the part of the vagina, as the rapid eversion of the cirris was always accompanied by a peculiar jerky movement of the genitalia visible in outline. According to Gamble (1897) and Faust (1939), self-fertilization of each proglottid is the usual procedure in cestodes, but cross-fertilization from one worm to another or from one proglottid to another in the same worm is not an infrequent occurrence. With the vertical erection of the cirris, it is difficult to see how self-fertilization of a proglottid can take place since the male and female genital pores are separate, and it seems likely that cross-fertilization must be the normal copulatory procedure in Schistocephalus. No copulation, however, was observed between two or three larvae cultured in the same tube, which suggests that copulation will only take place when the worms are in very close apposition, a condition presumably in which they find themselves in the alimentary canal of the bird host. Due to the hibernation of the fish during the winter months, it has not been possible to obtain additional material to test this hypothesis further.

From purely morphological grounds, the Ligulinae have long been regarded as ‘primitive’ cestodes. The fact that sexual maturity can be induced by the raising of the temperature in a suitable medium indicates that from the point of view of their physiology also, they are relatively unspecialized. This would account for their occurrence in a variety of final hosts. In most cyclophyllidean cestodes (e.g. Taenia saginata), on the other hand, there can be no question of the change in temperature being responsible for providing the stimulus for the transformation of the larval stage into the sexually mature strobila, as the intermediate hosts are also warm-blooded, so that other factors must operate. De Waele (1934) has suggested that the stimulus for the development of cysticercoids is provided by the digestive juices in the final host and that the cuticle of the cestodes serves to protect the worms from digestion, but only in the specific host or closely allied species. In veiw of this hypothesis, it is of interest to note that Joyeux & Baer (1942) obtained sexually mature strobila of Ligula by feeding the plerocercoids to ducks, gulls, dogs and cats, but failed to obtain development with rabbits, macaque monkeys and man. Since the body temperatures of these animals are not significantly different, it must be concluded that the physiological conditions of the juices of the gut also play some part in the development of the pseudophyllidean cestodes.

One of the most interesting features of the life cycle of Schistocephalus is that although its sexual development is virtually at a standstill when in the plerocercoid phase, it can nevertheless undergo considerable growth in body size. This is proved by the fact that larvae with 60–80 proglottids can be obtained from the fish. Why the somatic cells continue to grow, while the germ cells stop after a certain stage, is a question to which no satisfactory answer can as yet be given.

The present experiments have been relatively successful in the attainment of asepsis, prolonged viability and maturation of the genitalia in vitro, but it must be emphasized that they have been only preliminary and necessarily empirical in nature. Further work will be required to determine the factors concerned in the growth of the larval phase and the fertilization of the ova. The information and experience gained, however, will enable more extensive investigations to be undertaken with regard to many problems of paramount importance concerning the metabolism and general physiology of cestodes.

My thanks are due to Professor Spaul for advice and encouragement and to Dr Baylis for help with the literature.