Z. Parasitenk. 50, 313--321 (1976) 9 by Springer-Verlag 1976
Observations on the Histogenesis of Nervous Tissue in Diphyllobothrium dendriticum Nitzsch, 1824 (Cestoda, Pseudophyllidea) M a r g a r e t h a K. S. Gustafsson Institute of Biology, Abo Akademi, Abo, Finland lZeeeived February 4, 1976
Summary. The mode of growth of the populations of cells within and immediately surrounding the main lateral nerve cords in the actively growing, immature Diphyllobothrium dendriticum was studied by the use of aH-thymidine autoradiography. The population of nerve cells within the nerve cord grows only on account of cell migration from the surrounding parenchyma. No mitotic figures were observed in the nerve cords. The rate of growth is high. Within a period of cultivation for 2 days in hamster 34% of the nerve cells within the nerve cords have arrived from the parenchyma. These cells can be considered as cells at the starting point for differentiation into nerve cells. The protective layer of binding cells around the nerve cords also grows on account of cells migrating from the parenchyma. The binding cells actively move from the outer regions of the layer inwards close to the nerve cord. As stem cells for these types of cell differentiation serve the highly basophilic, actively dividing germinative cells. Introduction Since t h e l a t e 1800's a n d t h e e a r l y 1900's t h e n e r v o u s s y s t e m of cestodes has been s t u d i e d v e r y little. The o l d e r works have been r e v i e w e d b y H y m a n (1951), W a r d l e a n d McLeod (1952) a n d B u l l o c k a n d H o r r i g e (1965). I n t h e m a j o r i t y of r e c e n t i n v e s t i g a t i o n s h i s t o c h e m i c a l m e t h o d s h a v e b e e n used (Lee c t a l . , 1963; S c h a r d e i n a n d W a l t z , 1965; K r a l j , 1967; 0 h m a n - J a m e s , 1968; W i l s o n a n d Schiller, 1969; Shield, 1969, 1971). V e r y l i t t l e i n f o r m a t i o n a b o u t t h e cytol o g y of t h e n e r v o u s s y s t e m in cestodes is a v a i l a b l e . Morseth (1967) has d e s c r i b e d t h e u l t r a s t r u c t u r e of t h e n e r v o u s s y s t e m of Echinococcus granulosus. B y w a y of i n t r o d u c t i o n t h e n e r v o u s s y s t e m of Acanthobothrium coronatum a n d its c y t o l o g y is briefly d e s c r i b e d here (Rees a n d W i l l i a m s , 1965; Rees, 1966). I n A. coronatum t h e n e r v o u s s y s t e m consists of a b r a i n c o m p o s e d of two b i l o b e d ganglia j o i n e d b y a t r a n s v e r s e a n d a d o r s a l a n d v e n t r a l commissure. T e n longi t u d i n a l n e r v e cords rise from t h e b r a i n a n d e x t e n d t h r o u g h t h e scolex a n d t h e strobila. T h e cerebral ganglia do n o t c o n t a i n ganglion cells. S p i n d l e - s h a p e d ganglion cells are, however, f o u n d in t h e t r a n s v e r s e commissure a n d s c a t t e r e d bipolar n e r v e cells occur in t h e l o n g i t u d i n a l n e r v e cords. M u l t i p o l a r nerve cells occur o u t s i d e a n d in close association w i t h t h e nerve cords. B i n d i n g cells form a t h i n p r o t e c t i n g l a y e r a r o u n d t h e l o n g i t u d i n a l nerve cords. To m y k n o w l e d g e n o t h i n g is k n o w n a b o u t t h e histogenesis of nervous tissue in cestodes. The n e c k region of t h e a c t i v e l y growing i m m a t u r e a d u l t Diphyllobothrium dendriticum is c h a r a c t e r i z e d b y v e r y active cell p r o l i f e r a t i o n a n d histo-
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Margaretha K. S. Gustafsson
genesis. Cell proliferation is due e n t i r e l y to the division of the highly basophilic g e r m i n a t i v e cells. These constitute a pool, from which cells are d r a w n for diff e r e n t i a t i o n to various specialized cells (Wikgren, 1967 ; W i k g r c n a n d K n u t s , 1970; W i k g r e n a n d Gustafsson, 1971; W i k g r e n et al., 1971). The growth of the cell p o p u l a t i o n within a n d i m m e d i a t e l y s u r r o u n d i n g the m a i n lateral nerve cords is the object of this study. Special i n t e r e s t has been focused on the r e c r u i t m e n t of new n e r v e cells w i t h i n the nerve cords. The problem was s t u d i e d b y the use of 3 H - t h y m i d i n e autoradiography. Materials a n d Hethods PIeroeercoids of D. dendriticum were obtained from whitefish (Coregonus h~varetus) from lake Pyh~ji~rvi in SW Finland. Two series of experiments were performed. I. Cultivation in vivo. Plerocercoids were incubated at 37~ C for 4 h in Hanks' solution containing 0.5 ~c/ml aH-thymidine (sp. act. 5 c/mM), and given to 15 golden hamsters. Worms were collected after 44, 67, 86 and 92 h in the hamsters. II. Cultivation in vitro. 24 sterile plerocercoids were incubated at 37~C for 4 h in Medium 199 containing 0.5 ~zc/mlaH-thymidine (sp. act. 5 c/mS[), and then cultivated in aH-thymidine free medium for 20 and 40 h. 8 plerocercoids were fixed directly after incubation in aH-thymidine. Fixation was performed in one part neutral formalin in ten parts 70% alcohol. Paraffin sections were cut at 5 [zm. Part of the sections were stained with basic fuchsin according to Feulgen and prepared for autoradiography by coating with Kodak AR l0 stripping film. Some of the sections were stained with pyronine-methyl green after development. The exposure time was 8 days.
Results Nerve Cells within the M a i n Lateral Nerve Cords
The two m a i n lateral nerve cords i n the neck region of D. dendriticum measure a p p r o x i m a t e l y 30 • 20 [zm in cross section. T h e y consist of a b u n d l e of u n m y e l i n a t e d n e r v e fibres r u n n i n g r o u g h l y parallel to each other. Occasionally cell bodies can be seen lying b e t w e e n the n e r v e fibres. These cells are t e r m e d nerve cells on account of their location. According to Rees (1966) spindle-shaped nerve cells are present in the l o n g i t u d i n a l n e r v e cords in the strobila of A . eoronatum. The cytological characteristics a n d the exact n a t u r e of these cells will be dealt with i n a special report. There occur on average 7 nerve cells per 100 ~zm n e r v e cord. The cells are u n e v e n l y d i s t r i b u t e d b u t n o special t r e n d of periodicity has been observed. The s t a r t i n g p o i n t of this s t u d y is presented i n F i g u r e 1, which shows the d i s t r i b u t i o n of labelled cells in plerocercoids fixed i m m e d i a t e l y after 4 h i n c u b a t i o n i n aH-thymidine. I n t e r e s t is here focused on the p a r t of the i n n e r p ~ r e n c h y m a (IP) i m m e d i a t e l y s u r r o u n d i n g the m a i n lateral nerve cords, where a p p r o x i m a t e l y 36 % of the t o t a l cell p o p u l a t i o n has b e e n labelled. I n Table 1 the results of the two series of e x p e r i m e n t s are presented. F r o m the table it is e v i d e n t t h a t a l r e a d y differentiated n e r v e cells do n o t have the capacity to incorporate 3H-thymidinc. No labelled nerve cells were f o u n d in worms fixed directly after t r e a t m e n t with 8H-thymidine. However, after c u l t i v a t i o n for only 20 h in vitro labelled cells begin to appear w i t h i n the n e r v e cords. This t r e n d continues u n t i l 33.6% of the cells within the n e r v e cords are labelled i n
Histogenesis of Nervous Tissue in D. dendriticum
315
Fig. 1. Autoradiograph showing the distribution of labelled cells in a plerocercoid of Diphyllobothrium dendriticum fixed directly after incubation for 4 h in Medium 199 containing 3H-thymidine. • 100 Fig. 2. Cross section of the main lateral nerve cord of D. dendriticum reared in a hamster for 44 h. Note the heavily labelled cell within the nerve cord alongside unlabelled one. Pyronine-methyl green stain. • 1,300
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Margaretha K. S. Gustafsson
Table 1. The occurrence of labelled cells within the main lateral nerve cords of Diphyllobothrium dendriticum during cultivation in vitro and in vivo Cultivation conditions 4 h in aH-thymidine 20 h in vitro 40 h in vitro 44 h in vivo 67 h in vivo 86 h in vivo 92 h in vivo
No. of worms studied
No. of sections studied
Total no. of cells in the nerve cords observed
No. of labelled cells
% of labelled cells
8
180
55
0
0
12 5 8 3 2 2
240 248 648 378 165 159
104 54 274 109 44 40
23 18 92 29 9 9
22.1 33.3 33.6 26.6 20.5 22.5
worms r e a r e d in h a m s t e r s for 44 h after t h e i n i t i a l labelling with a H - t h y m i d i n e (Fig. 2). W i t h longer c u l t i v a t i o n in vivo t h e p r o p o r t i o n of l a b e l l e d nerve cells decreases to some e x t e n t b u t still persists a t 22.5% when t h e e x p e r i m e n t was finished after 92 h in vivo. I t should be m e n t i o n e d here t h a t m i t o t i c figures h a v e n e v e r been seen w i t h i n t h e nerve cords. This m e a n s t h a t t h e increase of l a b e l l e d cells w i t h i n the nerve cords c a n n o t be due to t h e division of ones a l r e a d y labelled.
The Layer o/Binding Cells The m a i n l a t e r a l nerve cords are s u r r o u n d e d b y a n i n c o m p l e t e l a y e r of cells t e r m e d binding cells. The n a m e originates from T o w e r ' s i n v e s t i g a t i o n of t h e nervous s y s t e m of Moniezia expansa (1900). T h e l a y e r of b i n d i n g cells is n o t s h a r p l y d e l i m i t e d from t h e s u r r o u n d i n g p a r e n c h y m a . Most of t h e b i n d i n g cells are s i t u a t e d on t h e l a t e r a l surface of t h e n e r v e cords, t h e r e f o r m i n g a r a t h e r c o m p a c t l a y e r of cells u s u a l l y two to t h r e e cells thick. On t h e i n n e r surface of t h e nerve cord facing t h e m a i n e x c r e t o r y d u c t s a n d on t h e d o r s a l a n d v e n t r a l surfaces v e r y few b i n d i n g cells can be seen. The b i n d i n g cells are c h a r a c t e r i z e d b y v e r y little, s l i g h t l y basophilic cytoplasm. T h e cells are v e r y small. The r o u n d t o oval nucleus measures 4.0-6.7 • 2.6-4.7 ~zm. The c y t o p l a s m of t h e b i n d i n g cells close to t h e nerve cord is often d r a w n out i n t o two processes p a r t l y s u r r o u n d i n g t h e n e r v e cord. The b i n d i n g cells a t t h e b o r d e r of t h e p a r e n c h y m a have s l i g h t l y more basophilic c y t o p l a s m t h a n those close t o t h e cord. The m o d e of g r o w t h of t h e l a y e r of b i n d i n g cells a r o u n d t h e n e r v e cord in t h e neck region of t h e a c t i v e l y growing D. dendriticum is e v i d e n t from T a b l e 2, which shows t h e result of in v i t r o c u l t i v a t i o n of worms p r e v i o u s l y t r e a t e d with 3H-thymidine. F r o m t h e t a b l e it a p p e a r s t h a t t h e p r o p o r t i o n of l a b e l l e d cells in t h e l a y e r of b i n d i n g cells rises from 4.6% in worms fixed i m m e d i a t e l y after inc u b a t i o n for 4 h in a H - t h y m i d i n e to 18.0% a n d 25,0% a f t e r s u b s e q u e n t cultiv a t i o n for 20 a n d 40 h respectively. The rise is considerable a n d is also e v i d e n t from F i g u r e s 3 a n d 4. I n a d d i t i o n to t h e quite obvious rise in t h e p r o p o r t i o n of l a b e l l e d b i n d i n g cells a r o u n d t h e n e r v e cord changes also t a k e place in t h e l o c a t i o n of these cells. F i g u r e 5 is a d i a g r a m of t h e n e r v e cord a n d its l a y e r of b i n d i n g cells. The l a y e r
Histogenesis of Nervous Tissue in D. dendriticum
317
Table 2. The occurrence of labelled cells in the layer of binding cells around the main lateral nerve cords of Diphyllobothrium dendriticum during cultivation in vitro Cultivation conditions
No. of worms studied
No. of cells in the layer of binding cells observed
No. of labelled cells in the layer of binding cells
% of labelled cells in the layer of binding cells
4 h in aH-thymidine 20 h in vitro 40 h in vitro
8
3622
168
4.6
8 5
2714 1503
488 375
18.0 25.0
of binding cells is divided into five regions. Figure 5 and Table 3 show the differences in the location of labelled cells in the layer of binding cells in worms fixed directly after incubation in aH-thymidine and after subsequent cultivation for 40 h. The changes are very clear. The nmnber of sections lacking labelled cells decreases considerably during cultivation. The number of labelled cells per section increases greatly. Furthermore, the labelled cells move inwards from region I to regions I I and I I I . Mitotic figures have not been observed in the layer of binding cells though they occur h'equently in the surrounding parenchyma. Discussion Nerve Cells within the M a i n Lateral Nerve Cords
Using autoradiographic techniques it was possible to follow the histogenesis of nervous tissue elements in the neck region of immature adult D. dendritieum. The results illustrate some interesting points regarding cell dynamics in worms during the early phases of infection in hamsters and during short-term cultivation in vitro. The results clearly indicate that the population of nerve cells within the nerve cords grows only on account of cell migration from the surrounding parenchyma. No mitotic divisions were observed within the nerve cords. According to Wikgren and Gustafsson (1971) the only cells capable of DNA-synthesis (i.e. incorporation of aH-thymidine) and division in the neck region of D. dendriticum are the germinative cells. Figure 1 shows the distribution of labelled germinative cells. The germinative cells thus actively migrate into the nerve cords thereby supplying cells for further differentiation into nerve cells. The rapid rise in the proportion of labelled nerve cells after only 20 h cultivation in vitro indicates very active cell migration and consequently also very intense growth of the cell population within the nerve cord. During the period between 20 and 40 h of in vitro cultivation the rate of accumulation of labelled nerve cells is somewhat lower than during the first 20 h. However, this need not mean that the growth of the population of cells within the nerve cords is considerably slower. The initial pool of labelled germinative cells is constantly being " d i l u t e d " because of repeated mitotic divisions. This means that the amount of aH-thymidine decreases up the point where the label of the nuclei are no longer distinguishable against the background. This dilution might also explain the decreasing values for labelled nerve cells accompanying longer cultivation in
318
Margaretha K. S. Gustafsson
Fig. 3. Cross s~etion of the main lateral nerve cord of .D. dendritieum fixed directly Mter incubation for 4 h in Medium 199 containing 3H-thymidine. No labelled cells occur in the layer of binding cells. Pyronine-methyl green stain. • 1,300 Fig. 4. Cross section of the main lateral nerve cord of D. dendriticura fixed after cultivation for 40 h after initial labelling with 3tI-thymidine. Note the large number of labelled cells in the layer of binding cells close to the nerve cord. Pyronine-melbhyl green stain. • 1,300
vivo. According to W i k g r c n a n d Gustafsson (1967) m i t o t i c a c t i v i t y is high during t h e first hours of c u l t i v a t i o n in vitro. The dura, tion of one mitotic cycle is app r o x i m a t e l y 19 h (Wikgren a n d Gustafsson, 1967). This m e a n s t h a t the germ i n a t i v e cells divide a p p r o x i m a t e l y once a day. According to B r s (1966)
Histogenesis of Nervous Tissue in D. dendriticum 4 hours
in
40 hours
3H-thymidine 21% 0 13 % ~)
I II
0 % (D
Ill
18 % e 48 %
@
vation
vc}
O~)~O~~c)
iv
I%
0
52 % Q) 21% Q 8 %
culti-
in v i t r o
18 % e
v
319
@
I II Ill IV v
:C." @
@
@
Fig. 5. Diagram showing the main nerve cord and its surrounding layer of binding cells in D. dendriticum. The layer of binding cells is divided into five regions. The left half of the picture shows the percentuM distribution of labelled cells in worms fixed directly after incubation in aH-thymidine. The right half shows the distribution of labelled cells after subsequent cultivation for 40 h in vitro Table 3. The occurrence of labelled cells in the layer of binding cells around the nerve cords of Diphyllobothrium dendriticum fixed directly after incubation for 4 h in aH-thymidine and after subsequent cultivation for 40 h in vitro
No. of worms studied No. of sections/worm studied Total no. of sections studied 57o. of sections lacking labelled cells % of sections lacking labelled cells Total no of labelled binding cells Mean no. of labelled binding cells/section
Incubated 4 h in 3H-thymidine
Cultivated 40 h in vitro
8 20 160 82 51 139 0.9
5 20 100 5 5 423 4.2
the growth of D. norwegicum ( n o w = D. dendritieum B y l u n d , 1969) is e x p o n e n t i a l b e t w e e n the first a n d the fifth d a y of infection in hamsters. The p o p u l a t i o n of cells w i t h i n the m a i n lateral n e r v e cords t h u s contains a fairly large p r o p o r t i o n of cells which have r e c e n t l y m i g r a t e d from the s u r r o u n d i n g p a r e n c h y m a a n d which can therefore be considered as cells at the s t a r t i n g p o i n t for differentiation into nerve cells. E M studies m i g h t reveal i n t e r e s t i n g i n f o r m a t i o n a b o u t these early stages of differentiation.
The Layer o/Binding Cells B i n d i n g cells have been described from A. coronatum, i n the neck region of which they form a loose, protective layer of cells on the outer border of the m a i n lateral nerve cords (Rees, 1966). F u r t h e r down i n the worm, i n more m a t u r e regions, the b i n d i n g cells become scattered a n d less a b u n d a n t . According to Rees (1966) the b i n d i n g cells do n o t divide at a rate which keeps in pace with the growth of the rest of the strobila. No data a b o u t mitotic divisions of b i n d i n g cells are given, however. A loose layer of b i n d i n g cells a r o u n d the nerve cords has further been reported from plerocercoids of Triaenophorus nodulosus (Gustafs-
320
Margaretha K. S. Gustafsson
son, 1973) a n d from a d u l t Echinococcus granulosus (Gustafsson, in press). No d a t a a b o u t t h e g r o w t h of t h e p o p u l a t i o n of these cells are a v a i l a b l e . The result of t h i s s t u d y shows t h a t t h e p o p u l a t i o n of b i n d i n g cells a r o u n d the nerve cords in D. dendriticum grows on account of g e r m i n a t i v e cells a c t i v e l y m i g r a t i n g t o w a r d s t h e nerve cords. As soon as t h e g e r m i n a t i v e cells reach t h e o u t e r m o s t regions of t h e l a y e r of b i n d i n g cells t h e y a p p a r e n t l y lose t h e i r a b i l i t y to d i v i d e m i t o t i c a l l y . F u r t h e r m o r e , on t h e i r w a y i n w a r d s t o w a r d s t h e surface of t h e nerve cord t h e a m o u n t of b a s o p h i l i a decreases m a r k e d l y . The few (less t h a n 5 %) l a b e l l e d cells i n c l u d e d in t h e l a y e r of b i n d i n g cells in worms fixed d i r e c t l y after i n c u b a t i o n in 3 H - t h y m i d i n c can be e x p l a i n e d b y t h e difficulties in d r a w i n g t h e e x a c t limits of t h e l a y e r of b i n d i n g cells. The labelled ceils in question can therefore be r e g a r d e d as belonging to t h e surrounding p a r c n c h y m a . The m i g r a t i o n of g e r m i n a t i v e cells i n t o t h e n e r v e cords a n d i n t o the l a y e r of binding cells is a similar p h e n o m e n o n to t h e m i g r a t i o n of g e r m i n a t i v e cells i n t o t h e l a y e r of t e g u m e n t M ceils in D. dendriticum ( W i k g r e n a n d K n u t s , 1970). G e r m i n a t i v e cells h a v e f u r t h e r been shown to c o n t r i b u t e to t h e f o r m a t i o n of p r i m a r y a n l a g e n b y m i g r a t i n g t o discrete loci in t h e i n n e r p a r e n c h y m a of D. dendriticum (Wikgren et al., 1971). The results of t h i s s t u d y s u p p o r t t h e a s s u m p t i o n t h a t t h e g e r m i u a t i v e cells serve as stem cells for d i f f e r e n t i a t i o n along different lines in D. dendriticum. Acknowledgement. The experiment was originally designed for the investigation of the growth of the subtegumental tissue in D. dendriticum. However, the material proved to contain valuable information about the formation of other tissues as well. The author whishes to thank Professor B.-J. Wikgren and G.M. Knuts for the use of their material and for helpful discussions during the course of the study.
Key to Lettering of Figures 1 - - 5 E excretory duct I P inner parenchyma LM longitudinal muscle layer
N main lateral nerve cord OP outer parenchyma T tegument
References Br~ten, T. : Studies of the helminth fauna of Norway. VII. Growth, fecundity, and fertility of Diphyllobothrium norvegicum Vik, (Cestoda) in golden hamsters. Nytt Mag. Zool. 13, 39-51 (1966) Bullock, T. H., Horridge, G. A. : Structure and function in the nervous system of invertebrates, Vol. I. San Francisco: W. H. Freeman 1965 Bylund, G. B. : Experimental investigations on Diphyllobothrium dendriticum (~ D. norvegicum) from Northern Finland. In Swedish with English summary. Parasit. Inst. Soc. Sol. Fenn. Inform. 10, 3-17 (1969) Gustafsson, M. K. S. : The histology of the neck region of pleroeercoids of Triaenophorua nodulosua (Cestoda, Pseudophyllidea). Acta zool. fenn. 188, 1-16 (1973) Gustafsson, M. K. S. : Basic cell types in Echinococcua granuloaus. (In press) ttyman, L. H. : The invertebrates: Platyhelminthes and rhynchocoela. The acoelomate bilateria, Vol. II. New York-Toronto-London: McGraw-Hill Book Co. Inc. 1951 KrMj, N. : Morphologic and histochemical studies on the nervous system of tapeworms revealed by the eholinesterase method (Taenia hydatigena, Dipylidium caninum and Moniezia expansa). Vet. Arh. (Zagreb) 87, 277-286 (1967)
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Lee, D . L . , Rothman, A . H . , Sentura, J . B . : Esterases in Hymenolepis and Hydatigera. Exp. Parasit. 14, 285-295 (1963) Morseth, D. J. : Observations on the fine structure of the nervous system of Echinococcus granulosus. J. Parasit. 58, 492 500 (1967) Ohman-James, C. : Histochemical studies of the cestodc Diphyllobothrium dendriticum Nitzsch, 1824. Z. Parasitenk. 80, 40 56 (1968) Rees, G. : Nerve cells in Acanthobothrium coronatum (Rud.) (Cestoda: Tetraphyl]idea). Parasitology 56, 45-54 (1966) t~ecs, G., Williams, H . H . : The functional morphology of the scolex and the genitalia of Acanthobothrium coronatum (Rud.) (Ccstoda: Tetraphyllidea). Parasitology 55, 617-651 (1965) Schardein, J . L . , Waitz, J . A . : Histochemical studies of esterases in the cuticle and nerve cords of four cyclophyllidean cestodes. J. Parasit. 51, 356-363 (1965) Shield, J. M. : Dipylidium caninum, Echinococcus granulosus and Hydatigera taeni/ormis: Histochcmical identification of eholinesterases. Exp. Parasit. 25, 217-231 (1969) Shield, J. M. : Histochemical localization of monoamines in the nervous system of Dipylidium canium (Cestoda) by the formaldehyde flourescense technique. Int. J. Parasit.1, 135-138 (1971) Tower, W. L. : The nervous system in the cestode Moniezia expansa. Zool. Jb. Anat. 18, 359-384 (1900) Wardle, R. A., McLeod, J. A. : The zoology of tapeworms. Minneapolis: University Minnesota Press 1952 Wikgren, B.-J. P.: Aspects on the kinetics of the cell population in cestodes. In: H. Teir, T. l~yt6maa, eds., Control of cellular growth in adult organisms. :New York and London: Academic Press 1967 Wikgren, B.-J. P., Gustafsson, M. K. S. : Duration of the cell cycle of germinative cells in pleroccrcoids of Diphyllobothrium dendriticum. Z. Parasitenk. 29, 275-281 (1967) Wikgren, B.-J. P., Gustafsson, M. K. S. : Cell proliferation and histogenesis in diphyllobotriid tapeworms (Cestoda). Acta Acad. Aboensis (B) 81, 1-10 (1971) Wikgren, B.-J. P., Gustafsson, M. K. S., ](nuts, G. M. : Primary anlage formation in diphyllobithriid tapeworms. Z. Parasitenk. 86, 131-139 (1971) Wikgren, B.-J. P., Knuts, G.M.: Growth of the subtegumcntal tissue in cestodes by cell migration. Acta Acad. Aboensis (B) 80, 1-6 (1970) Wilson, V. C. L. C., Schiller, E. L. : The neuroanatomy of Hymenolepis diminuta and H. nana. J. Parasit. 55, 261-270 (1969) Phil. lic. Margaretha K. S. Gustafsson Institute of Biology, Abo Akademi Porthansgatan 3 SF-20500 Abo 50, Finland
Observations on the Histogenesis of Nervous Tissue in Diphyllobothrium dendriticum Nitzsch, 1824 (Cestoda, Pseudophyllidea) M a r g a r e t h a K. S. Gustafsson Institute of Biology, Abo Akademi, Abo, Finland lZeeeived February 4, 1976
Summary. The mode of growth of the populations of cells within and immediately surrounding the main lateral nerve cords in the actively growing, immature Diphyllobothrium dendriticum was studied by the use of aH-thymidine autoradiography. The population of nerve cells within the nerve cord grows only on account of cell migration from the surrounding parenchyma. No mitotic figures were observed in the nerve cords. The rate of growth is high. Within a period of cultivation for 2 days in hamster 34% of the nerve cells within the nerve cords have arrived from the parenchyma. These cells can be considered as cells at the starting point for differentiation into nerve cells. The protective layer of binding cells around the nerve cords also grows on account of cells migrating from the parenchyma. The binding cells actively move from the outer regions of the layer inwards close to the nerve cord. As stem cells for these types of cell differentiation serve the highly basophilic, actively dividing germinative cells. Introduction Since t h e l a t e 1800's a n d t h e e a r l y 1900's t h e n e r v o u s s y s t e m of cestodes has been s t u d i e d v e r y little. The o l d e r works have been r e v i e w e d b y H y m a n (1951), W a r d l e a n d McLeod (1952) a n d B u l l o c k a n d H o r r i g e (1965). I n t h e m a j o r i t y of r e c e n t i n v e s t i g a t i o n s h i s t o c h e m i c a l m e t h o d s h a v e b e e n used (Lee c t a l . , 1963; S c h a r d e i n a n d W a l t z , 1965; K r a l j , 1967; 0 h m a n - J a m e s , 1968; W i l s o n a n d Schiller, 1969; Shield, 1969, 1971). V e r y l i t t l e i n f o r m a t i o n a b o u t t h e cytol o g y of t h e n e r v o u s s y s t e m in cestodes is a v a i l a b l e . Morseth (1967) has d e s c r i b e d t h e u l t r a s t r u c t u r e of t h e n e r v o u s s y s t e m of Echinococcus granulosus. B y w a y of i n t r o d u c t i o n t h e n e r v o u s s y s t e m of Acanthobothrium coronatum a n d its c y t o l o g y is briefly d e s c r i b e d here (Rees a n d W i l l i a m s , 1965; Rees, 1966). I n A. coronatum t h e n e r v o u s s y s t e m consists of a b r a i n c o m p o s e d of two b i l o b e d ganglia j o i n e d b y a t r a n s v e r s e a n d a d o r s a l a n d v e n t r a l commissure. T e n longi t u d i n a l n e r v e cords rise from t h e b r a i n a n d e x t e n d t h r o u g h t h e scolex a n d t h e strobila. T h e cerebral ganglia do n o t c o n t a i n ganglion cells. S p i n d l e - s h a p e d ganglion cells are, however, f o u n d in t h e t r a n s v e r s e commissure a n d s c a t t e r e d bipolar n e r v e cells occur in t h e l o n g i t u d i n a l n e r v e cords. M u l t i p o l a r nerve cells occur o u t s i d e a n d in close association w i t h t h e nerve cords. B i n d i n g cells form a t h i n p r o t e c t i n g l a y e r a r o u n d t h e l o n g i t u d i n a l nerve cords. To m y k n o w l e d g e n o t h i n g is k n o w n a b o u t t h e histogenesis of nervous tissue in cestodes. The n e c k region of t h e a c t i v e l y growing i m m a t u r e a d u l t Diphyllobothrium dendriticum is c h a r a c t e r i z e d b y v e r y active cell p r o l i f e r a t i o n a n d histo-
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Margaretha K. S. Gustafsson
genesis. Cell proliferation is due e n t i r e l y to the division of the highly basophilic g e r m i n a t i v e cells. These constitute a pool, from which cells are d r a w n for diff e r e n t i a t i o n to various specialized cells (Wikgren, 1967 ; W i k g r c n a n d K n u t s , 1970; W i k g r e n a n d Gustafsson, 1971; W i k g r e n et al., 1971). The growth of the cell p o p u l a t i o n within a n d i m m e d i a t e l y s u r r o u n d i n g the m a i n lateral nerve cords is the object of this study. Special i n t e r e s t has been focused on the r e c r u i t m e n t of new n e r v e cells w i t h i n the nerve cords. The problem was s t u d i e d b y the use of 3 H - t h y m i d i n e autoradiography. Materials a n d Hethods PIeroeercoids of D. dendriticum were obtained from whitefish (Coregonus h~varetus) from lake Pyh~ji~rvi in SW Finland. Two series of experiments were performed. I. Cultivation in vivo. Plerocercoids were incubated at 37~ C for 4 h in Hanks' solution containing 0.5 ~c/ml aH-thymidine (sp. act. 5 c/mM), and given to 15 golden hamsters. Worms were collected after 44, 67, 86 and 92 h in the hamsters. II. Cultivation in vitro. 24 sterile plerocercoids were incubated at 37~C for 4 h in Medium 199 containing 0.5 ~zc/mlaH-thymidine (sp. act. 5 c/mS[), and then cultivated in aH-thymidine free medium for 20 and 40 h. 8 plerocercoids were fixed directly after incubation in aH-thymidine. Fixation was performed in one part neutral formalin in ten parts 70% alcohol. Paraffin sections were cut at 5 [zm. Part of the sections were stained with basic fuchsin according to Feulgen and prepared for autoradiography by coating with Kodak AR l0 stripping film. Some of the sections were stained with pyronine-methyl green after development. The exposure time was 8 days.
Results Nerve Cells within the M a i n Lateral Nerve Cords
The two m a i n lateral nerve cords i n the neck region of D. dendriticum measure a p p r o x i m a t e l y 30 • 20 [zm in cross section. T h e y consist of a b u n d l e of u n m y e l i n a t e d n e r v e fibres r u n n i n g r o u g h l y parallel to each other. Occasionally cell bodies can be seen lying b e t w e e n the n e r v e fibres. These cells are t e r m e d nerve cells on account of their location. According to Rees (1966) spindle-shaped nerve cells are present in the l o n g i t u d i n a l n e r v e cords in the strobila of A . eoronatum. The cytological characteristics a n d the exact n a t u r e of these cells will be dealt with i n a special report. There occur on average 7 nerve cells per 100 ~zm n e r v e cord. The cells are u n e v e n l y d i s t r i b u t e d b u t n o special t r e n d of periodicity has been observed. The s t a r t i n g p o i n t of this s t u d y is presented i n F i g u r e 1, which shows the d i s t r i b u t i o n of labelled cells in plerocercoids fixed i m m e d i a t e l y after 4 h i n c u b a t i o n i n aH-thymidine. I n t e r e s t is here focused on the p a r t of the i n n e r p ~ r e n c h y m a (IP) i m m e d i a t e l y s u r r o u n d i n g the m a i n lateral nerve cords, where a p p r o x i m a t e l y 36 % of the t o t a l cell p o p u l a t i o n has b e e n labelled. I n Table 1 the results of the two series of e x p e r i m e n t s are presented. F r o m the table it is e v i d e n t t h a t a l r e a d y differentiated n e r v e cells do n o t have the capacity to incorporate 3H-thymidinc. No labelled nerve cells were f o u n d in worms fixed directly after t r e a t m e n t with 8H-thymidine. However, after c u l t i v a t i o n for only 20 h in vitro labelled cells begin to appear w i t h i n the n e r v e cords. This t r e n d continues u n t i l 33.6% of the cells within the n e r v e cords are labelled i n
Histogenesis of Nervous Tissue in D. dendriticum
315
Fig. 1. Autoradiograph showing the distribution of labelled cells in a plerocercoid of Diphyllobothrium dendriticum fixed directly after incubation for 4 h in Medium 199 containing 3H-thymidine. • 100 Fig. 2. Cross section of the main lateral nerve cord of D. dendriticum reared in a hamster for 44 h. Note the heavily labelled cell within the nerve cord alongside unlabelled one. Pyronine-methyl green stain. • 1,300
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Margaretha K. S. Gustafsson
Table 1. The occurrence of labelled cells within the main lateral nerve cords of Diphyllobothrium dendriticum during cultivation in vitro and in vivo Cultivation conditions 4 h in aH-thymidine 20 h in vitro 40 h in vitro 44 h in vivo 67 h in vivo 86 h in vivo 92 h in vivo
No. of worms studied
No. of sections studied
Total no. of cells in the nerve cords observed
No. of labelled cells
% of labelled cells
8
180
55
0
0
12 5 8 3 2 2
240 248 648 378 165 159
104 54 274 109 44 40
23 18 92 29 9 9
22.1 33.3 33.6 26.6 20.5 22.5
worms r e a r e d in h a m s t e r s for 44 h after t h e i n i t i a l labelling with a H - t h y m i d i n e (Fig. 2). W i t h longer c u l t i v a t i o n in vivo t h e p r o p o r t i o n of l a b e l l e d nerve cells decreases to some e x t e n t b u t still persists a t 22.5% when t h e e x p e r i m e n t was finished after 92 h in vivo. I t should be m e n t i o n e d here t h a t m i t o t i c figures h a v e n e v e r been seen w i t h i n t h e nerve cords. This m e a n s t h a t t h e increase of l a b e l l e d cells w i t h i n the nerve cords c a n n o t be due to t h e division of ones a l r e a d y labelled.
The Layer o/Binding Cells The m a i n l a t e r a l nerve cords are s u r r o u n d e d b y a n i n c o m p l e t e l a y e r of cells t e r m e d binding cells. The n a m e originates from T o w e r ' s i n v e s t i g a t i o n of t h e nervous s y s t e m of Moniezia expansa (1900). T h e l a y e r of b i n d i n g cells is n o t s h a r p l y d e l i m i t e d from t h e s u r r o u n d i n g p a r e n c h y m a . Most of t h e b i n d i n g cells are s i t u a t e d on t h e l a t e r a l surface of t h e n e r v e cords, t h e r e f o r m i n g a r a t h e r c o m p a c t l a y e r of cells u s u a l l y two to t h r e e cells thick. On t h e i n n e r surface of t h e nerve cord facing t h e m a i n e x c r e t o r y d u c t s a n d on t h e d o r s a l a n d v e n t r a l surfaces v e r y few b i n d i n g cells can be seen. The b i n d i n g cells are c h a r a c t e r i z e d b y v e r y little, s l i g h t l y basophilic cytoplasm. T h e cells are v e r y small. The r o u n d t o oval nucleus measures 4.0-6.7 • 2.6-4.7 ~zm. The c y t o p l a s m of t h e b i n d i n g cells close to t h e nerve cord is often d r a w n out i n t o two processes p a r t l y s u r r o u n d i n g t h e n e r v e cord. The b i n d i n g cells a t t h e b o r d e r of t h e p a r e n c h y m a have s l i g h t l y more basophilic c y t o p l a s m t h a n those close t o t h e cord. The m o d e of g r o w t h of t h e l a y e r of b i n d i n g cells a r o u n d t h e n e r v e cord in t h e neck region of t h e a c t i v e l y growing D. dendriticum is e v i d e n t from T a b l e 2, which shows t h e result of in v i t r o c u l t i v a t i o n of worms p r e v i o u s l y t r e a t e d with 3H-thymidine. F r o m t h e t a b l e it a p p e a r s t h a t t h e p r o p o r t i o n of l a b e l l e d cells in t h e l a y e r of b i n d i n g cells rises from 4.6% in worms fixed i m m e d i a t e l y after inc u b a t i o n for 4 h in a H - t h y m i d i n e to 18.0% a n d 25,0% a f t e r s u b s e q u e n t cultiv a t i o n for 20 a n d 40 h respectively. The rise is considerable a n d is also e v i d e n t from F i g u r e s 3 a n d 4. I n a d d i t i o n to t h e quite obvious rise in t h e p r o p o r t i o n of l a b e l l e d b i n d i n g cells a r o u n d t h e n e r v e cord changes also t a k e place in t h e l o c a t i o n of these cells. F i g u r e 5 is a d i a g r a m of t h e n e r v e cord a n d its l a y e r of b i n d i n g cells. The l a y e r
Histogenesis of Nervous Tissue in D. dendriticum
317
Table 2. The occurrence of labelled cells in the layer of binding cells around the main lateral nerve cords of Diphyllobothrium dendriticum during cultivation in vitro Cultivation conditions
No. of worms studied
No. of cells in the layer of binding cells observed
No. of labelled cells in the layer of binding cells
% of labelled cells in the layer of binding cells
4 h in aH-thymidine 20 h in vitro 40 h in vitro
8
3622
168
4.6
8 5
2714 1503
488 375
18.0 25.0
of binding cells is divided into five regions. Figure 5 and Table 3 show the differences in the location of labelled cells in the layer of binding cells in worms fixed directly after incubation in aH-thymidine and after subsequent cultivation for 40 h. The changes are very clear. The nmnber of sections lacking labelled cells decreases considerably during cultivation. The number of labelled cells per section increases greatly. Furthermore, the labelled cells move inwards from region I to regions I I and I I I . Mitotic figures have not been observed in the layer of binding cells though they occur h'equently in the surrounding parenchyma. Discussion Nerve Cells within the M a i n Lateral Nerve Cords
Using autoradiographic techniques it was possible to follow the histogenesis of nervous tissue elements in the neck region of immature adult D. dendritieum. The results illustrate some interesting points regarding cell dynamics in worms during the early phases of infection in hamsters and during short-term cultivation in vitro. The results clearly indicate that the population of nerve cells within the nerve cords grows only on account of cell migration from the surrounding parenchyma. No mitotic divisions were observed within the nerve cords. According to Wikgren and Gustafsson (1971) the only cells capable of DNA-synthesis (i.e. incorporation of aH-thymidine) and division in the neck region of D. dendriticum are the germinative cells. Figure 1 shows the distribution of labelled germinative cells. The germinative cells thus actively migrate into the nerve cords thereby supplying cells for further differentiation into nerve cells. The rapid rise in the proportion of labelled nerve cells after only 20 h cultivation in vitro indicates very active cell migration and consequently also very intense growth of the cell population within the nerve cord. During the period between 20 and 40 h of in vitro cultivation the rate of accumulation of labelled nerve cells is somewhat lower than during the first 20 h. However, this need not mean that the growth of the population of cells within the nerve cords is considerably slower. The initial pool of labelled germinative cells is constantly being " d i l u t e d " because of repeated mitotic divisions. This means that the amount of aH-thymidine decreases up the point where the label of the nuclei are no longer distinguishable against the background. This dilution might also explain the decreasing values for labelled nerve cells accompanying longer cultivation in
318
Margaretha K. S. Gustafsson
Fig. 3. Cross s~etion of the main lateral nerve cord of .D. dendritieum fixed directly Mter incubation for 4 h in Medium 199 containing 3H-thymidine. No labelled cells occur in the layer of binding cells. Pyronine-methyl green stain. • 1,300 Fig. 4. Cross section of the main lateral nerve cord of D. dendriticura fixed after cultivation for 40 h after initial labelling with 3tI-thymidine. Note the large number of labelled cells in the layer of binding cells close to the nerve cord. Pyronine-melbhyl green stain. • 1,300
vivo. According to W i k g r c n a n d Gustafsson (1967) m i t o t i c a c t i v i t y is high during t h e first hours of c u l t i v a t i o n in vitro. The dura, tion of one mitotic cycle is app r o x i m a t e l y 19 h (Wikgren a n d Gustafsson, 1967). This m e a n s t h a t the germ i n a t i v e cells divide a p p r o x i m a t e l y once a day. According to B r s (1966)
Histogenesis of Nervous Tissue in D. dendriticum 4 hours
in
40 hours
3H-thymidine 21% 0 13 % ~)
I II
0 % (D
Ill
18 % e 48 %
@
vation
vc}
O~)~O~~c)
iv
I%
0
52 % Q) 21% Q 8 %
culti-
in v i t r o
18 % e
v
319
@
I II Ill IV v
:C." @
@
@
Fig. 5. Diagram showing the main nerve cord and its surrounding layer of binding cells in D. dendriticum. The layer of binding cells is divided into five regions. The left half of the picture shows the percentuM distribution of labelled cells in worms fixed directly after incubation in aH-thymidine. The right half shows the distribution of labelled cells after subsequent cultivation for 40 h in vitro Table 3. The occurrence of labelled cells in the layer of binding cells around the nerve cords of Diphyllobothrium dendriticum fixed directly after incubation for 4 h in aH-thymidine and after subsequent cultivation for 40 h in vitro
No. of worms studied No. of sections/worm studied Total no. of sections studied 57o. of sections lacking labelled cells % of sections lacking labelled cells Total no of labelled binding cells Mean no. of labelled binding cells/section
Incubated 4 h in 3H-thymidine
Cultivated 40 h in vitro
8 20 160 82 51 139 0.9
5 20 100 5 5 423 4.2
the growth of D. norwegicum ( n o w = D. dendritieum B y l u n d , 1969) is e x p o n e n t i a l b e t w e e n the first a n d the fifth d a y of infection in hamsters. The p o p u l a t i o n of cells w i t h i n the m a i n lateral n e r v e cords t h u s contains a fairly large p r o p o r t i o n of cells which have r e c e n t l y m i g r a t e d from the s u r r o u n d i n g p a r e n c h y m a a n d which can therefore be considered as cells at the s t a r t i n g p o i n t for differentiation into nerve cells. E M studies m i g h t reveal i n t e r e s t i n g i n f o r m a t i o n a b o u t these early stages of differentiation.
The Layer o/Binding Cells B i n d i n g cells have been described from A. coronatum, i n the neck region of which they form a loose, protective layer of cells on the outer border of the m a i n lateral nerve cords (Rees, 1966). F u r t h e r down i n the worm, i n more m a t u r e regions, the b i n d i n g cells become scattered a n d less a b u n d a n t . According to Rees (1966) the b i n d i n g cells do n o t divide at a rate which keeps in pace with the growth of the rest of the strobila. No data a b o u t mitotic divisions of b i n d i n g cells are given, however. A loose layer of b i n d i n g cells a r o u n d the nerve cords has further been reported from plerocercoids of Triaenophorus nodulosus (Gustafs-
320
Margaretha K. S. Gustafsson
son, 1973) a n d from a d u l t Echinococcus granulosus (Gustafsson, in press). No d a t a a b o u t t h e g r o w t h of t h e p o p u l a t i o n of these cells are a v a i l a b l e . The result of t h i s s t u d y shows t h a t t h e p o p u l a t i o n of b i n d i n g cells a r o u n d the nerve cords in D. dendriticum grows on account of g e r m i n a t i v e cells a c t i v e l y m i g r a t i n g t o w a r d s t h e nerve cords. As soon as t h e g e r m i n a t i v e cells reach t h e o u t e r m o s t regions of t h e l a y e r of b i n d i n g cells t h e y a p p a r e n t l y lose t h e i r a b i l i t y to d i v i d e m i t o t i c a l l y . F u r t h e r m o r e , on t h e i r w a y i n w a r d s t o w a r d s t h e surface of t h e nerve cord t h e a m o u n t of b a s o p h i l i a decreases m a r k e d l y . The few (less t h a n 5 %) l a b e l l e d cells i n c l u d e d in t h e l a y e r of b i n d i n g cells in worms fixed d i r e c t l y after i n c u b a t i o n in 3 H - t h y m i d i n c can be e x p l a i n e d b y t h e difficulties in d r a w i n g t h e e x a c t limits of t h e l a y e r of b i n d i n g cells. The labelled ceils in question can therefore be r e g a r d e d as belonging to t h e surrounding p a r c n c h y m a . The m i g r a t i o n of g e r m i n a t i v e cells i n t o t h e n e r v e cords a n d i n t o the l a y e r of binding cells is a similar p h e n o m e n o n to t h e m i g r a t i o n of g e r m i n a t i v e cells i n t o t h e l a y e r of t e g u m e n t M ceils in D. dendriticum ( W i k g r e n a n d K n u t s , 1970). G e r m i n a t i v e cells h a v e f u r t h e r been shown to c o n t r i b u t e to t h e f o r m a t i o n of p r i m a r y a n l a g e n b y m i g r a t i n g t o discrete loci in t h e i n n e r p a r e n c h y m a of D. dendriticum (Wikgren et al., 1971). The results of t h i s s t u d y s u p p o r t t h e a s s u m p t i o n t h a t t h e g e r m i u a t i v e cells serve as stem cells for d i f f e r e n t i a t i o n along different lines in D. dendriticum. Acknowledgement. The experiment was originally designed for the investigation of the growth of the subtegumental tissue in D. dendriticum. However, the material proved to contain valuable information about the formation of other tissues as well. The author whishes to thank Professor B.-J. Wikgren and G.M. Knuts for the use of their material and for helpful discussions during the course of the study.
Key to Lettering of Figures 1 - - 5 E excretory duct I P inner parenchyma LM longitudinal muscle layer
N main lateral nerve cord OP outer parenchyma T tegument
References Br~ten, T. : Studies of the helminth fauna of Norway. VII. Growth, fecundity, and fertility of Diphyllobothrium norvegicum Vik, (Cestoda) in golden hamsters. Nytt Mag. Zool. 13, 39-51 (1966) Bullock, T. H., Horridge, G. A. : Structure and function in the nervous system of invertebrates, Vol. I. San Francisco: W. H. Freeman 1965 Bylund, G. B. : Experimental investigations on Diphyllobothrium dendriticum (~ D. norvegicum) from Northern Finland. In Swedish with English summary. Parasit. Inst. Soc. Sol. Fenn. Inform. 10, 3-17 (1969) Gustafsson, M. K. S. : The histology of the neck region of pleroeercoids of Triaenophorua nodulosua (Cestoda, Pseudophyllidea). Acta zool. fenn. 188, 1-16 (1973) Gustafsson, M. K. S. : Basic cell types in Echinococcua granuloaus. (In press) ttyman, L. H. : The invertebrates: Platyhelminthes and rhynchocoela. The acoelomate bilateria, Vol. II. New York-Toronto-London: McGraw-Hill Book Co. Inc. 1951 KrMj, N. : Morphologic and histochemical studies on the nervous system of tapeworms revealed by the eholinesterase method (Taenia hydatigena, Dipylidium caninum and Moniezia expansa). Vet. Arh. (Zagreb) 87, 277-286 (1967)
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