International Journal

for Parasitology.

1976. Vol. 6. pp. 143-149. Per.gamon Press. Printed in Great Britain.

STUDIES ON THE IN VITRO CULTURE OF

TAENIA

CRASSZCEPS

GERALD W. ESCH~ and J. D. SMYTH~ ‘Department of Biology, Wake Forest University, Winston-Salem, N.C. 27109, U.S.A., and 2Department Zoology, Imperial College of Science and Technology, University of London, London, England

of

(Received 6 February 1975) Abstract-Esm

G. W. and SMYTH J. D. 1976. Studies on the m vitro culture of Taenia crassiceps. International Journalfor Parasitology 6: 143-149. Taenia crassiceps (KBS and Toi strains) were grown in vitro to the strobilar stage. The culture system consisted of 100 parts of medium “858”, 25 parts of heat-inactivated fetal calf serum and 12.5 parts of 5 % yeast extract; the medium was enriched by the addition of glucose, KCI, penicillin and streptomycin. The liquid phase was overlaid upon a solid base (coagulated bovine serum) and was gassed by addition of 10% O2 + 5 % CO, in N, or 10% CO, in N,. Following appropriate treatment with acid-pepsin and pancreatin-trypsin-sodium taurocholate, stimulated cysticerci were introduced into culture vessels in a shaking water bath at 38°C. Evidence of morphogenesis was obvious within 24-48 h, proglottis formation occurred within 7 days and by 13 days, genital pores had formed. Testes were observed to develop to the 64-cell stage. Subsequent experiments, employing this culture technique, revealed that pretreatment with enzymes and/or bile salts was not essential to in vitro growth and development of T. crussiceps. It was found, however, that T. crassiceps requires the presence of the serum globulin fraction in fetal calf serum in order for any kind of morphogenesis to occur. The results of these studies are compared to those reported for in vitro culture of Echinococcus granulosus.

INDEX KEY WORDS: Cestoda; Tueniu crassiceps; growth and development; calf serum; immunoglobulins; serum growth factor.

INTRODUCTION

ATTEMPTS to develop in vitro culture

methods for taeniid cestodes from post-embryonic larval stages to the adult have, with the exception of Echinococcus grunulosus (Smyth, 1967), not been made (Silverman, 1965; Silverman & Hansen, 1971). The present report describes the partially successful efforts to culture Taenia crassiceps and compares its requirements for growth and development with those of E. grunulosus (Smyth, 1967). MATERIALS

AND

METHODS

Parasite. Cysticerci of Taeniu crassiceps, maintained by the serial subinoculation method of Freeman (1962), were used in the present studies. Attempts were made to culture the normal (or wild-type) KBS and Toi strains as well as the mutant ORF strain (Smith, Esch & Kuhn, 1972); for details on the isolation and maintenance of the strains, see Dorais & Esch (1969) and Freeman (1962). Culture system. The liquid phase consisted of 100 parts of medium “858” (Difco), 25 parts of heat inactivated (30 min at 56°C) fetal calf serum (Grand Island Biological Supply), and 12.5 parts of 5% powdered yeast extract (Oxoid or Grand Island Biological Supply). To a liter of this medium was added 22 ml of 30% glucose, 5.0 ml of 2.0% KCI, 100 units/ml penicillin G and 100 pg/ml streptomycin sulphate. The resulting medium (designated hereafter as Medium V) was found to be

organogenesis;

fetal

effective in terms of supporting satisfactory growth and development of T. crussiceps. In several experiments, IPT-fetal calf serum was substituted for heat inactivated fetal calf serum; the IPT-fetal calf serum was certifiedfree of all immunoprecipitins (immunoglobulins) by Grand Island Biological Supply, from whom it was purchased. The worms were grown in 30 ml capacity plastic culture flasks containing 10 ml of Medium V. A solid base consisted of 5.0 ml of coagulated bovine serum (Oxoid or Grand Island Biological Supply). The serum was coagulated at 75°C for 60-90 min; the consistency of the base was such that enzyme-bile salt activated cysticerci were able to penetrate and actively migrate without becoming entrapped. In some experiments, the base was broken into small pieces with a sterile Pasteur pipette; this system, too, was satisfactory in supporting growth and development of cysticerci. The gas phase in the culture flasks was either 10% 0, + 5% CO, in N, or 10% CO, in N,. Both were satisfactory in terms of supporting the worms during culture experiments. Culture protocol. The methods employed were basically those of Smyth (1967) which were originally developed for Echinoco>&granulosus, although these have since been modified (Smvth & Davies, 1974). Larval T. c&.&eps were removed aseptically from infected mice killed previously by cervical dislocation. The larvae were placed into Petri dishes containing warm (38.9”C) Hank’s balanced salt solution. A 25 ml aliquot, containing several dozen cysticerci, was removed to a

143

GERALD W. ETCH and J. D.

144

Falcon flask, and the larvae washed twice in two volume. of warm Hank’s solution. Following the second wash. 50 ml of Hank’s solution with 10 mg/ml pepsin (BDH), pH 1.9, was added to the flask and left for 20 min during which time the flask was constantly agitated in a waterbath at 38*9”C. The larvae were then washed 3 times in 25 ml of warm Hank’s solution and transferred to a flask containing an evaginating solution of Hank’s with 3 mg/ml pancreatin (BDH), 0.1 mg/ml sodium taurocholate (NBC) and 0.15 mg/ml trypsin (BDH). The larvae were kept in a shaking water bath at 38.9”C for between three and 20 h. After washing twice in warm Hank’s solution, the activated larvae were transferred to a Petri dish containing warm Medium V. At this point, larvae were examined with an inverted microscope in order to determine which would be

utilized in the culture experiments. Only those larvae which were evaginated and whose scoleces had well developed hooks (mature individuals), were selected ; these cysticerci generally exhibited “trumpeting” or “hook raising” movements (see Results section for description). The stimulated larvae were then transferred to flasks containing the appropriate liquid and solid phases. Usually, 3-6 larvae were added to each flask which was then gassed and the experiment begun. Culture flasks, maintained in a shaking water bath at 38_9”C, (dog body temperature) were examined periodically with an inverted microscope kept in a constant temperature chamber at 38.9’C. The liquid phase was changed, as necessary (usually every 48 h), in a laminar flow sterile air hood to minimize contamination. When cont~ination did occur, tetracycline hydr~hloride or neomycin sulfate was added, without apparent harm to the worms and with varying degrees of success. RESULTS Evugination. Upon exposure to pepsin, larvae did not appear (overtly) to change in respect to the magnitude of the rhythmic contraction of the bladder wall, nor did the scoleces or rostella exhibit behavior different from that of untreated larvae. However, treatment with pancreatin-taurocholateTABLE I-TIMINGOF

SMYTH

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6. 1976

trypsin (p-t-t) elicited an explosive response within 5 min after initial exposure. The initial reaction to p-t-t began with alternating contraction and extension (trumpeting) in the neck region, separating the bladder from the scolex. In time, the neck became greatly elongated, with the larvae increasing in length 2 or 3-fold. Shortly after trumpeting began, the scolex and rostellum were also affected; the suckers became quite active, exhibiting what could be described as a “searching” type of behavior. Simultaneously, the rostellum evaginated and the anteriorly pointing hook blades in non-stimulated larvae, were directed posteriorly. The change in direction of the blades was accompanied by a flattening of the entire rostellar region. The activity of the hooks and rostellum will hereafter be described as “hook raising”. Throughout the duration of exposure to p-t-t, trumpeting and hook raising persisted. When the enzymes and bile salts were removed and the activated larvae transferred to culture flasks, these activities continued, but at a less frenetic pace. Several experiments were conducted in which bile salts alone were used to stimulate rostellar evagination. In these instances, growth and development did take place although there was a 3-5 day lag in developmental time when compared to that which occurred after complete (p-t-t) treatment. In several experiments, cultures were begun with cysticerci which had been removed from mice, washed in Hank’s BSS, incubated 3 h in the same solution (without benefit of enzyme or bile salt pretreatment) and transferred to culture flasks. In each of these experiments, larvae did not display trumpeting or hook raising behavior. However, growth and development proceeded without any sort of enzyme-bile salt treatment, to the same stage as larvae which had been given the full p-t-t treatment. As shown in Table 1, the onset in development of untreated cysticerci was delayed up to 13 days; CONDITIONS

T. ~~~~~~~~~~GRO~HANDDEVELOPME~~~~ERDIFFERE~

-

Stage number

1 2 3 4 2 7

Description of stage

Neck elongation Osmoregulatory canals prominent Fluting of canals Banding Froglottid~tion Cirrus streak formation Genital pore fo~ation

“Experiment terminated at IS dlys. j-X.0. = not observed.

Ex~rimen~i protocol and earliest time (in days) required to reach described stage A C D* B Full enzyme-bile No pepsin or Full enzyme-bile No pepsin salt treatment pretreatment; pancreatin-bile salt treatment followed by otherwise salt treatment; except Med. V. culture in otherwise as in A made with Medium V as in A FCS-IPT -~ 1 3 4 1 I 4 3 N.0.I 2 3 4 7 13

6 7 8 15 16 _~._________

4 5 6 19 26

N.O. N.O. N.O. N.O. N.O. ~._.-___“..._.-

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in vitro culture of Taenia crussiceps

145

Stage I nonetheless, these larvae grew to the same stage of morphogenesis as any p-t-t treated larvae. The Neck elongation experiments were repeated (I 6 ffasks with neariy 100 Day I larvae were involved) and each time, the results were the same. The full pepsin, p-t-t treatment was variable in effect on the bladder wall; in some cases, the bIadder collapsed, but in no case was there evidence of digestion as had been reported for Taenia pisiformis (Smyth, 1967). Cursory observations suggested the bladders of older cysticerci were more prone to collapse than younger larvae and buds. After introduction of activated cysticerci into culture flasks, either (1) the bladder remained attached to the strobila throughout the developmental sequence, or (2) the bladder was physically disengaged from the strobila during migration in the base, or (3) the bladder was detached when terminal proglottids were first shed after approximately 10 days of culture. Stage 1: Neck elongation (Figs. 1 and 3). On introduction of activated cysticerci into culture vessels, larvae generally penetrated into the coagulated bovine serum base. This was accomplished by entrance via holes punched in the base, or dire&y, without benefit of holes. Movement into the base was effected through the coordinated use of suckers and rosteilar hooks; migration continued for several days. During the initial 24 h of culture, the first signs of morphogenesis were observed; inevitably, this FIG. 1. Stage 1, showing a cysticercus during the first involved substantial elongation of the neck (two to 24 h foIi~wing enzyme-bile salt treatment. three times the previous length). It is questionable whether this was due to generation of new tissue. More likely, it was the result of stretching and Stage 4: Banding (Figs. 2 and 3). As the transrelaxation of preexisting tissue produced during Iucence moved toward the bladder, vertical condevelopment of the cysticercus while present in the solidation occurred in the darkened area between abdominal cavity of the mouse. the canals. The result was the formation of a Stage 2: Appearance of osmoregulatory canals distinctively “banded” strobila. (Figs. 2 and 3). Foliowing neck elongation, and Stage 5: ~rog~ottis for~tion (Figs. 2 and 3). during the first 24 hours of culture, the previously Within 96 h of introduction into culture vessels, the indistinct osmoregulatory canals became strikingly light areas separating the bands in the neck had apparent. The heightened visual appearance of the reorganized to the extent that interproglottid septa canals seemed to be related to a change in the were recognizable. Proglottis formation was more refractiveness of the neck and scoiex tegument pronouns in areas distal to the scolex, as might be which became more transparent. The tegumental expected. border became virtually cIear, a characteristic which Stage 6: Civvus streak formation (see Figs. 2 and 3). persisted as long as the worm remained in a healthy The first reproductive structure to be seen clearly condition. When the tegument became translucent was the cirrus streak. It developed as a delicate, and grey, there was usually a rapid decline in the band of cells which extended from the midline, overall condition of the worm. laterally in the proglottis. It was usually observed in Stage 3: Fluting of canais (Figs. 2 and 3). Within the anterior third of the most posterior proglottides the first 4g h of development, the OsmoreguIatory of the strobila. canafs became fluted. The fluting began posterior to Stage 7: Genitalpore for~tio~ (see Figs. 2 and 3). the scolex and extended toward the bladder as neck By day 13, a fully formed cirrus and cirrus pouch elongation proceeded. Simultaneously, the area were clearly seen to terminate in a genital atrium between the canals, immediately posterior to the which opened and closed in an irregular rhythm. scolex, became translucent. As fluting and neck examination of fixed and stained specimens revealed growth continued, the translucence moved prothe presence of both the vagina and sperm duct, gressively toward the bladder. leading from the medullary parenchyma to the

146

GERALD

ESCH

Stage 3

Stage 2 Osmoregulatory canals promment Day

W.

Fluting

I

of

canals

Day 2

Stage 4

Stage 5

Stage 6

Bandlng

Proglottidlzatlon

Cirrus

Day 3

Day 4

Day 7

FIG. 2. Stages 2 through 6, showing the nature of growth and development of culture. Stage

I

I

Osmoregulatory Day

3

4

5

1

1 7

canals prominent

I

Fluting

of COnOIS

Day 2

Banding Day 3

Proglottiditation Day 4

6

1976

streak

through the first 7 days

terminal genital structures. Internally, testes, in various stages of development, were observed. After 11 days of culture, testes were seen for the first time;

Neck elongation Day

I.J.P. VOL. 6.

and J. D. SMYTH

Cirrus streak Day 7

Genital Dare Day 13

FIG. 3. A composite of the primary features of growth and

development through 13 days of culture.

aceto-orcein squashes revealed testes in the 8- and 16-cell stages while &cell stages were observed in later cultures. Female reproductive organs were difficult to identify in living specimens or in acetoorcein squashes. However, masses of cells, in areas where one might expect vitellaria and ovaries to develop, were seen in whole mount preparations. Sperm were, at no time, noted. Several cultures were extended for as long as 80 days but failed to mature beyond the point described above. Coagulated bovine serum base. The coagulated bovine serum base was essential for support of growth and deveIopment of Taenia crassiceps through Stage 7. Worms cultured without bases failed to develop beyond Stage 5 (proglottis formation). Even then, the worms were in poor condition, i.e. heavily vesiculated, and died very rapidly. When agar was used in place of bovine serum, vesiculation and death of the worms occurred within 96 h after ~ginning the experiment. FetaE calf serum re~aireme~ts. Growth and development of the worms was rapid and proceeded with less vesiculation and mortality when the fetal calf serum was heat-inactivated at 56°C for 30 min. Additional, carefully controlled, experiments would have to be conducted in order to confirm this observation. Frequently, scoleces were observed

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6. 1976

with an “immune precipitate” extruding from the rostellar region. Such a “precipitate” was frequently present at the time the larvae were moved from the mouse abdominal cavity. It was usually retained after enzyme-bile salt pretreatment and, in some cases, grew in size during culture. The precipitate was not seen in association with larvae which exhibited scolex trumpeting or hook raising. While the exact nature of the precipitate is not known, it is possible that it resulted from an initial in vivo immunological reaction. Increase in the size of the precipitate in vitro, would suggest that fetal calf serum contained non-specific antibodies capable of reacting with parasite antigens. During the course of evaluating the basic experimental protocol, a series of experiments were designed in which fetal calf serum, minus the immunoprecipitin fraction, (FCS-IPT) was used instead of normal fetal calf serum. All elements of the previously described basic experimental protocol were used in these experiments. Worms cultured in this manner failed to develop beyond Stage 1 (Table 1). This experiment was repeated several times except that larvae from the same population were divided following enzyme-bile salt pretreatment. Half were added to culture vessels containing Medium V made with FCS-IPT and the other half to Medium V prepared with normal fetal calf serum. The results were identical to those of the initial experiment; FCS-IPT in Medium V did not support development beyond Stage 1 while the other worms grew to Stage 7 in normal time. It is clear that an TABLE

2---SUMMARY

Liquid phase

OF SEVERAL

*FCS+YE+“858”

Pretreatment protocol Pepsin-Nat-

FCS+YE+“858”

pancreatin-trypsin Nat-pancreatin

FCS+YE+“858” FCS+YTLE+“858” FCS+YP+“858” FCS+YE+“858” FCS-IEYT+YE+“858” FCS+YE+“858” FCS+YE+“858” FCS+YE+“858” FCS+YE+“858”

Pepsin-Natpancreatin Pepsin-Natpancreatin-trypsin Pepsin-Natpancreatin-trypsin Pepsin-Natpancreatin-trypsin Pepsin-Natpancreatin-trypsin Non-Stimulated Pepsin-Natpancreatin-trypsin Pepsin (5 %)-Natpancreatin-trypsin Pepsin (1 %)-Natpancreatin-trypsin

147

element(s) contained in the immunoprecipitin fraction is necessary for strobilization to occur. A summary of this experiment, plus others in which the basic protocol was varied, are shown in Table 2. Strains of T. crassiceps. Throughout the above experiments, the Toi and ORF strains (Freeman, 1962), or the KBS strain (Dorais & Esch, 1969) were used. The only basic difference between the results obtained using Toi and KBS larvae was that the timing of proglottis formation was about 24 h earlier in the former strain. When larvae of the ORF strain were exposed to the pp-t treatment, there was absolutely no behavioral response nor was there any growth during subsequent culture in Medium V. We assume this is due to the absence of a scolex in ORF larvae which, in turn, reflects the aneuploid condition of the strain (Smith, Esch & Kuhn, 1972). DISCUSSION A successful technique for the in vitro culture of Echinococcus granulosus protoscoleces to the adult

stage was described by Smyth (1967) and Smyth & Davies (1974). After pepsin treatment protoscoleces were evaginated with pancreatin-trypsin-taurocholate or taurocholate alone and grown in a diphasic system to the point that sperm development occurred; insemination did not occur, suggesting either (a) the absence of a factor or factors necessary to complete maturation or (b) the mechanical failure of insemination due to the lack of strobilar compressions or related physical factors in the

EXPERIMENTS

IN WHICH

THE BASIC PROTOCOL*

WAS VARIED

Segmentation Comments time (days) 4 Diphasic medium. Gas phase aerobic. Genital 6 7 5 9 A

not observed 6

pore in 13 days. Diphasic medium. Gas phase aerobic. Genital pore in 16 days. Monophasic medium. Gas phase aerobic. All larvae vesiculated and moribund after 13 days. Monophasic medium. Gas phase aerobic. All larvae vesiculated and moribund after 13 days. Monophasic medium. Gas phase aerobic. All larvae vesiculated and moribund after 13 days. Diphasic medium. Gas phase aerobic. Genital pore in 19 days. Diphasic medium. Gas phase anaerobic. Diphasic medium. pore in 26 days. Diphasic medium. pore in 14 days. Diphasic medium. pore in 26 days. Diphasic medium. pore in 19 days.

Gas phase anaerobic. Genital Gas phase anaerobic. Genital Gas phase aerobic. Genital Gas phase aerobic. Genital

Note: FCS = fecal calf serum; YE = yeast extract; Nat = sodium taurocholate; diphasic medium = with coagulated bovine serum base and liquid phase; YTLE = yeastolate (Oxoid); YP = yeast paste (Oxoid); aerobic = 85% N,-10% 02-5% COz; anaerobic = 90% N, + 10% CO,.

148

GERALD

W. ESCH and J. D. SMYTH

culture system. The procedure described herein for Taenia crassiceps is similar to that devised for E. granulosus except that development of T. crassiceps stopped short of complete sexual maturation. That the culture requirements for the two species are similar is not surprising, since both utilize canines as the definitive host. These are however, a number of differences between the culture requirements of the two species. First, a comparison of in vivo developmental times by T. crassiceps and E. granulosus indicate that both species require approximately 40 days in order for egg production to occur (Freeman, 1962; Smyth, 1967). On the other hand, E. granulosus was shown to require 14 days for segmentation in vitro (Smyth, 1967) while T. crassiceps required only 4 days. Second, Smyth (1967) reported that a proteinaceous base was necessary for segmentation and subsequent development by E. granulosus. Apparently, this is only a partial requirement by T. crassiceps since proglottis formation will occur when activated larvae are grown in a monophasic medium. However, development of T. crassiceps in a monophasic medium always culminated in vesiculation, and death, within 13 days. In Smyth’s system (1967) vesiculation of E. granulosus in a monophasic system did occur, but usually in 2C22 days. In our hands an agar base was completely unsatisfactory in supporting any form of development by either species. An important difference between E. granulosus and T. crassiceps is that the protoscolex of the former is in an unevaginated condition on removal from the hydatid cyst while the cysticercus of the latter is frequently evaginated. According to Smyth (1967) E. granulosus must be evaginated (and activated) by suitable enzyme-bile salt pretreatment, followed by subsequent culture on a suitable base in order to effect strobilization. The scoleces of most mature T. crassiceps, on the other hand, are already evaginated on removal from the intermediate host. It is obvious therefore that these cysticerci do not require enzyme-bile salt pretreatment for scolex evagination. In order to induce rostellar evagination, activate scolex trumpeting and cause hook raising, the enzyme-bile salt treatment is, however, necessary, even though this pretreatment is not a prerequisite for strobilization or organogenesis in T. crassiceps.

A number of studies (Buecher, Hansen & Gottfried, 1969; Buecher, Hansen & Gottfried, 1970; Hansen & Berntzen, 1969), indicate that specific proteins, i.e. a, fi or y-globulins, are essential constituents for successful culture of several parasitic and free-living nematodes and one tapeworm Hymenolepis nana. The present study has shown that the immunoprecipitin fraction (which includes a, f3 and y-globulins) is, or contains a component which is, necessary for growth and development of Taenia crassiceps beyond Stage 1. Such a finding is

I.J.P. VOL. 6.

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not surprising, especially in view of the well known observations on the effects of a serum growth factor in stimulating cell growth during in vitro culture of some tissues (Jainchill & Todaro, 1970; Todaro, Maysuya, Bloom, Robbins & Green, 1967). The latter authors reported that the growth promoting factor migrates with gamma globulins on Sephadex G-200 and is non-dialyzable. According to Jainchill & Todaro (1970), the growth factor is not gamma globulin itself since both agamma calf serum and human agammaglobulinemic serum are potent stimulators of in vitro cell growth. In the previously mentioned parasite systems and in the case of Taenia crassiceps, identity of the growth stimulating factor has not been made. In all of the previous studies of which we are aware (for reviews see Smyth, 1969; Silverman & Hansen, 1971), there is a clear indication that for taeniid cestodes, enzyme and/or bile salt treatment is necessary for scolex and/or rostellar evagination and that this event(s) is a necessary forerunner of strobilization and organogenesis. Our studies with T. crassiceps do not parallel the previous observations. They indicate that evagination and subsequent morphogenesis are distinct events, i.e. rostellar evagination can be stimulated by enzymebile salt treatment while growth and development (strobilization and organogenesis) can occur without benefit of such stimulation. In view of these observations, it is suggested that scolex and/or rostellar evagination are probably necessary for attachment in a canine’s intestine and that subsequent growth is effected when and if another factor is present to initiate and sustain strobilization and organogenesis. While the nature of the necessary stimulus remains to be elucidated, it would seem reasonable to suggest that it may be associated with the serum growth factor previously mentioned. This assertion would necessarily assume that such a factor, or a related one, is a normal constituent of the intestinal milieu and that it would be released by cells of the intestinal mucosa or via the liver in the bile. Such speculation would also require that its’ action be blocked while cysticerci are present within the abdominal cavity of the intermediate host. In Hymenolepis microstoma, Seidel (1971) has shown that hemin is an essential factor for strobilization but we have no evidence, as yet, of such a requirement for T. crassiceps. It must be stressed that these experiments are very much of a preliminary nature and much more work is required before consistent reproducible results can be obtained. For example, different batches of serum, bile and T. crassiceps larvae gave somewhat inconsistent results-a phenomenon well recognized and allowed for in both tissue culture and parasite culture systems. This question has been discussed in some detail by Smyth & Davies (1974). Nevertheless, the development of the in vitro culture system for T. crussiceps-incomplete as it is-could

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In vilvo culture of Taenia crassiceps

provide a useful research tool for the study of developmental phenomena in tapeworms. The observation that worms grow rapidly in culture (strobilating in 96 h), that they will develop to the cirrus sac stage, and that they are relatively large compared to cyclophyllidean tapeworms used in other culture systems, combine to make T. crassiceps a useful organism for use in studies on developmental physiology of cestodes. Acknowledgements-me authors wish to thank Wake Forest University for their generous financial assistance during the leave-of-absence of the senior author. We also want to thank Mr. Jack Chernin for providing the stock infections of the Toi strain of Taenia crassiceps. This work was supported in part by a World Health Organization Research Fellowship awarded to G.W.E. for study in the laboratory of Professor Smyth and by a grant from the Wake Forest University Research and Publication Fund. The work was also supported in part by the U.K. Medical Research Council and the U.S. Atomic Energy Commission (under Contract AT38-l(301) between the University of Georgia and the U.S. Atomic Energy Commission). Contribution No. 284 from the W. K. Kellogg Biological Station, Hickory Corners, Michigan.

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FREEMANR. S. 1962. Studies on the biology of Tuenia crassieeps (Zeder, 1800), Rud., 1810 (Cestoda). Canadian Journal of Zoology 40: 969-990. HANSENE. L. & BERNTZENA. K. 1969. Development of Caenorhabditis briggsae and Hymenolepis nnna in interchanged media. Journal of Parasitology 55: 1012-1017. JAINCHILLJ. L. & TODAROG. J. 1970. Stimulation of cell growth in vitro by serum with and without growth factor. Exoerimental Ceil Research 59: 137-l 46. SEIDELJ. S. i971. Hemin as a requirement in the development in vitro of Hymenolepis microstoma (Cestoda: Cyclophyllidea). Journey of Parasitoiogy 48: 551-554. SILVERMANP. H. 1965. In vitro culture procedures for parasitic helminths. In Advances in Pur~s~fo~ogy (Edited by BEN DAWES), Vol. 3, pp. 159-222. Academic Press, London. SILVERMAN P. H. & HANSEN E. L. 1971. In vitro cultivation procedures for parasitic helminths: recent advances. In Advances in Parasitology (Edited by BEN DAWES),Vol. 9, pp. 227-258. Academic Press, London. SMITH J. K., ESCH G. W. & KUHN R. E. 1972. Growth and development of larval Taenia crassiceps (Cestoda). 1. Aneupioidy in the anomalous (ORF) strain. Znternational JournaE for Purasiioiogy 2: 261-263. SUYTH J. D. 1967. Studies on tapeworm physiology. XI. in vitro culture of Erh~nococcus gr~nuiosus from the protoscolex to the strobilate stage. Pffrasj6o/ogy 57: Ill-133. SMYTHJ. D. 1969. The PhysioZogy of Cestodes. W. H. Freeman, San Francisco. SMYTH J. D. & DAVIESZ. 1974. In vitro culture of the strobilar stage of Echinococcus grunulosus (sheep strain): a review of basic problems and results. Znternational Journal for Parasitology 4: 631-644. TODAROG. J., MAYSUYAT., BLOOMS., ROBBINSA. & GREEN H. 1967. Growth Reguhtting Substances for Animaf Cells in Culture (Edited by DEFENDI V.- & STOKER M.). Wistar Institute Symposium No. 7, p. 87. Wistar, Philadelphia.