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Ultrastructural and histochemical studies of the differentiating plerocercoid cuticle of Schistocephalus… Morris, Gerald Patrick 1966

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ULTRASTRUCTURAL AND HISTOCHEMICAL  STUDIES  OF  THE DIFFERENTIATING PLEROCERCOID CUTICLE OF SCHISTOCEPHALUS  SOLIDUS.  ;  by GERALD PATRICK MORRIS B . S c . ( H o n s ) , The U n i v e r s i t y o f B r i t i s h C o l u m b i a ,  A THESIS SUBMITTED I N PARTIAL FULFILMENT  OF  THE REQUIREMENTS FOR THE DEGREE OF MASTER OF  SCIENCE  i n the Department of ZOOLOGY We a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e required standard  THE U N I V E R S I T Y OF B R I T I S H COLUMBIA JUNE,  1966  1964  In p r e s e n t i n g requirements Columbia, for  the  Head o f my  understood cial  gain  that shall  Department of  the  study.  copying of  this  Library  thesis  Department or  copying or not  be  the  shall  by  his  University  of  make i t f r e e l y  publication  of  Columbia  this  available  permission  thesis  written  the  British  representatives.  a l l o w e d w i t h o u t my  1966  f u l f i l m e n t of  f o r s c h o l a r l y p u r p o s e s may  Zoology  J u n e 28,  in p a r t i a l  I f u r t h e r agree that  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, C a n a d a  Date  thesis  advanced degree at  I agree that  r e f e r e n c e and  tensive by  f o r an  this  for be  exgranted  It is for  finan-  permission.  i  ABSTRACT  To date, most s t u d i e s o f the cestode c u t i c l e have been c a r r i e d o u t on a d u l t worms and i n no i n s t a n c e has an attempt been made t o c o r r e l a t e u l t r a s t r u c t u r a l and histochemical observations.  The present study was  to o b t a i n i n f o r m a t i o n on both the h i s t o c h e m i c a l  designed  composition  and the u l t r a s t r u c t u r e o f the p l e r o c e r c o i d c u t i c l e o f the p s e u d o p h y l l i d e a n cestode 1776)  S c h i s t o c e p h a l u s s o l i d u s (Mflller,  and t o study any changes which might occur  plerocercoid  during  differentiation.  Initially,  t h i s work has demonstrated t h a t the  p l e r o c e r c o i d tegument o f _S. s o l i d u s i s o f the same fundamental type as t h a t o f p r e v i o u s l y d e s c r i b e d c e s t o d e s .  An o u t e r  a n u c l e a t e b u t c e l l u l a r r e g i o n (the c u t i c l e ) i s connected by c y t o p l a s m i c tubes to n u c l e a t e d tegumental c e l l s l y i n g i n the parenchyma. I t has been shown t h a t the pore c a n a l s o f £>. s o l i d u s are d i f f e r e n t from any d e s c r i b e d f o r o t h e r cestodes i s suggested  on m o r p h o l o g i c a l  i n a secretory capacity.  and i t  grounds t h a t they may f u n c t i o n  In a d d i t i o n , the pore c a n a l s p r o v i d e  a d i r e c t c o n n e c t i o n between c e r t a i n parenchymal c e l l s and the e x t e r n a l medium. P a p i l l a - l i k e c u t i c u l a r processes on the c u t i c u l a r s u r f a c e have been d e s c r i b e d and both adhesive f u n c t i o n s are suggested  f o r these s t r u c t u r e s .  and a b s o r p t i v e I t has been  shown t h a t the c u t i c u l a r processes are not present  i n the  s m a l l e s t forms s t u d i e d but r a t h e r appear a t about the same time as the worm becomes i n f e c t i v e . Changes i n the h i s t o c h e m i c a l  and u l t r a s t r u e t u r a l  composition o f the c u t i c u l a r m a t r i x have been d e s c r i b e d .  It  i s probable t h a t i n c r e a s e d p l e r o c e r c o i d growth i s accompanied by a change i n c u t i c u l a r composition from a more proteinaceous s t a t e to one which i s l a r g e l y c a r b o h y d r a t e . A s u r f a c e sulphomucin-basic p r o t e i n complex has been d e s c r i b e d and p a r t i a l l y c h a r a c t e r i z e d .  P o s s i b l e f u n c t i o n s have  been suggested f o r t h i s l a y e r . RNA  as w e l l as a c i d and a l k a l i n e phosphatases were  found i n the c u t i c l e and the p o s s i b l e s i g n i f i c a n c e o f  these  substances i n c u t i c u l a r s y n t h e t i c a c t i v i t i e s i s c o n s i d e r e d . I t i s suggested t h a t p l e r o c e r c o i d c u t i c u l a r d i f f e r e n t i a t i o n may c o n s i s t o f two phases:  (1) an  phase i n which v a r i o u s mechanisms are e l a b o r a t e d s u r v i v a l i n the v e r t e b r a t e  i n t e s t i n e , and  i n which the p l e r o c e r c o i d i s m o d i f i e d growth i n the s t i c k l e b a c k coelom.  initial  to a l l o w  (2) a second phase  to permit prolonged  iii  TABLE OF CONTENTS PAGE INTRODUCTION  1  MATERIALS AND METHODS  4  A. . Source o f M a t e r i a l .  4  B.  P r e p a r a t i o n f o r E l e c t r o n Microscopy.  4  C.  P r e p a r a t i o n f o r L i g h t Microscopy.  5  D.  Preparation f o r Morphological  Studies.  5  E.  Preparation f o r Histochemical  Studies.  6  (1) (2) (3) (4) (5)  Tests Tests Tests Tests Tests  f o r carbohydrates f o r nucleic acids f o r enzymes f o r proteins for lipids  RESULTS OF LIGHT MICROSCOPY A.  Morphological (1) (2)  B.  10 Observations.  Fixation Morphology o f the d i f f e r e n t i a t i n g plerocercoid  Histochemical Results. (1) (2) (3) (4) (5)  Uistribution Distribution Distribution Distribution Distribution  of of of of of  (2) (3) (4)  10 10 10 14  carbohydrates nucleic acids enzymes proteins lipids  RESULTS OF ELECTRON MICROSCOPY (1)  6 7 7 8 8  Morphology o f d i f f e r e n t i a t e d plerocercoids Morphology o f u n d i f f e r e n t i a t e d plerocercoids Observations on embedding and f i x a t i o n techniques Observations on s t a i n i n g techniques  14 24 26 28 30 33 33 38 39 41  iv  PAGE DISCUSSION  42  SUMMARY  55  L I S T OF ABBREVIATIONS  57  PLATE I  59  (Figures  PLATE I I  (Figures  PLATE I I I PLATE I V  9-16)  (Figures (Figures  PLATE V ( F i g u r e s PLATE V I  1-8)  17-24) 25-29)  30-32)  ( F i g u r e s 33 a n d 34)  PLATE V I I PLATE V I I I  (Figures  35-37)  ( F i g u r e s 38 a n d 39)  60 61 62 63 64 65 66  LITERATURE CITED  67  APPENDIX A  74  APPENDIX B  75  V  ACKNOWLEDGEMENTS  I wish t o express my s i n c e r e thanks to Dr. C. V. Finnegan, n o t o n l y f o r h i s e x c e l l e n t support and advice, b u t a l s o f o r p e r m i t t i n g me t o choose my own area o f r e s e a r c h . I should  a l s o l i k e t o thank Dr. Finnegan f o r h i s w i l l i n g n e s s t o  spend many hours d i s c u s s i n g t o p i c s which are u s u a l l y l e f t to a helminthological for h i s c r i t i c a l  fate.  Dr. A. B. Acton r e c e i v e s my g r a t i t u d e  r e a d i n g o f my t h e s i s and f o r much  h e l p w i t h the e l e c t r o n microscopy.  appreciated  I am a l s o v e r y g r a t e f u l  to Mr. L. Veto f o r h i s a s s i s t a n c e w i t h the e l e c t r o n microscopy. Dr.  J . R. Adams i s to be thanked f o r h i s c r i t i c a l  reading of  my t h e s i s and f o r i n t r o d u c i n g me to the organism which forms the s u b j e c t o f t h i s d i s s e r t a t i o n . Mr.  I should  l i k e to thank  C. F. A. C u l l i n g f o r r e a d i n g my t h e s i s and f o r h i s  e x c e l l e n t comments on the h i s t o c h e m i s t r y .  Finally,  I should  l i k e t o thank Dr. R. N. Band f o r p r o v i d i n g a s t i m u l a t i n g introduction to b i o l o g i c a l  research.  1  INTRODUCTION  I t has  r e c e n t l y become apparent t h a t the  c u t i c l e i s not the n o n - c e l l u l a r s e c r e t e d  l a y e r of  cestode classical  d e s c r i p t i o n , but  r a t h e r i s p a r t of an a c t i v e l y m e t a b o l i z i n g  integument.  u l t r a s t r u c t u r a l s t u d i e s of Kent  The  Rothman (1963) and the cestode outer  (1962, 1965)  "tegument" c o n s i s t s o f two  anucleate  membrane and and  Threadgold  layer  possessing  (1957),  have shown t h a t  portions:  (1)  (the c u t i c l e ) bounded by a plasma numerous m i c r o v i l l u s - l i k e m i c r o t r i c h e s ,  (2) an i n n e r c e l l u l a r l a y e r j o i n e d to the outer  by numerous c y t o p l a s m i c separated  an  tubules.  These two  l a y e r s are  by an a c e l l u l a r s u b c u t i c u l a r membrane.  y e t c e r t a i n whether the outer  anucleate  I t i s not  p o r t i o n of  tegument i s s y n c y t i a l or compartmentalized  portion  the  (Threadgold,  1962,  1965). The  above s t u d i e s have p r o v i d e d  framework f o r the r e s u l t s of b i o c h e m i c a l , histochemical (see Read and  s t u d i e s on cestodes. Simmons, 1963)  a morphological p h y s i o l o g i c a l and  A number of workers  have demonstrated t h a t cestodes  possess the a b i l i t y to s e l e c t i v e l y absorb m a t e r i a l s environment and  s i n c e cestodes l a c k a l l t r a c e s o f a d i g e s t i v e  c a v i t y t h i s f u n c t i o n must be performed by the Histochemical and  Lee,  19 63;  from t h e i r  studies  (Bogitsh,  and Waitz and  1963;  Lee  Schardein,  presence i n the c u t i c l e of l i p i d s ,  cuticle.  et a l . , 1963;  1964)  protein,  Rothman  have shown the polysaccharides,  2  and such enzymes as a c i d phosphatase, a l k a l i n e phosphatase and those a s s o c i a t e d w i t h t e r m i n a l o x i d a t i o n . To date most s t u d i e s o f the c u t i c l e have been c a r r i e d out on a d u l t worms o f groups other than the pseudophyllideans and,  i n no case has an attempt been made to c o r r e l a t e  u l t r a s t r u c t u r a l and h i s t o c h e m i c a l data. was  designed  study  t o o b t a i n i n f o r m a t i o n on the h i s t o c h e m i c a l and  u l t r a s t r u c t u r a l composition  o f the d i f f e r e n t i a t i n g p l e r o c e r c o i d  c u t i c l e o f the p s e u d o p h y l l i d e a n (Mdller,  The p r e s e n t  cestode  Schistocephalus s o l i d u s  1776). The  growth o f the p l e r o c e r c o i d o f £>. s o l i d u s takes  p l a c e i n the second i n t e r m e d i a t e host, the t h r e e - s p i n e d stickleback  (Gasterosteus a c u l e a t u s ) .  The worm i s l o c a t e d i n  the coelomic c a v i t y and the weight o f p l e r o c e r c o i d s i n one f i s h may t o t a l up to 1/2 o r more o f the h o s t ' s body weight (Dogiel et, al_., 1961).  Somatic growth i s a p p a r e n t l y completed i n  the p l e r o c e r c o i d stage, which i s unique among cestodes i n t h a t i t i s completely segmented  (Smyth, 1946).  When the worm e n t e r s i t s f i n a l host, any one o f s e v e r a l species of piscivorous b i r d s , and i s passed  i t r a p i d l y reaches  i n the faeces w i t h i n 4-6 days.  sexual m a t u r i t y  An i n c r e a s e i n  temperature t o t h a t o f the b i r d h o s t i s the o n l y stimulus necessary t o induce d i f f e r e n t i a t i o n of the g e n i t a l i a 1946).  (Smyth,  The t r a n s i t i o n from the l a r v a l to the a d u l t form i s  accomplished  s o l e l y through  t i s s u e d i f f e r e n t i a t i o n which  i s supported b y endogenous r e s e r v e s accumulated d u r i n g  3  p l e r o c e r c o i d development  (Hopkins and McCaig, 1963).  The  complete l i f e c y c l e of S. s o l i d u s and the morphology o f the l a r v a l stages are d e s c r i b e d 1954).  i n Appendix A  (Taken from  Clarke,  4  MATERIALS  A.  and METHODS  Source o f M a t e r i a l .  P l e r o c e r c o i d s were o b t a i n e d from the coelom o f the t h r e e - s p i n e d s t i c k l e b a c k , Gasterosteus a c u l e a t u s , from l o c a l b r a c k i s h waters.  The s t i c k l e b a c k s were d e c a p i t a t e d  and the worms were removed immediately  from the h o s t ' s coelom  and weighed to the n e a r e s t .0005 gm on an e l e c t r i c  B.  taken  Preparation f o r Electron  balance.  Microscopy.  E n t i r e worms were p l a c e d i n Palade's  fixative  a t pH 7.2, 6% g l u t a r a l d e h y d e b u f f e r e d to pH 7.2, o r 4% paraformaldehyde  and s l i c e d i n t o t h i n segments w i t h a  razor  blade, b e f o r e b e i n g t r a n s f e r r e d to f r e s h f i x a t i v e f o r 1-2 hours.  Glutaraldehyde and paraformaldehyde-fixed  was p o s t - f i x e d i n osmium f o r 1% hours.  tissue  Tissue f i x e d i n  Palade's f i x a t i v e or g l u t a r a l d e h y d e was embedded i n Epon 812  (Luft, 1961) o r A r a l d i t e  i n paraformaldehyde  (Luft, 1961), w h i l e t h a t f i x e d  was embedded i n Maraglas  (Spurlock e t a l . ,  1963). S e c t i o n s were c u t w i t h g l a s s k n i v e s on a P o r t e r Blum ultramicrotome  and s t a i n e d w i t h l e a d c i t r a t e o r l e a d  a c e t a t e and u r a n y l a c e t a t e .  Photomicrographs were  taken  w i t h an H i t a c h i HU-11A and an H i t a c h i HS-7S a t m a g n i f i c a t i o n s of 2,000 to 100,000.  5  C.  Preparation  f o r L i g h t Microscopy.  The worms used f o r the m a j o r i t y o f the h i s t o c h e m i c a l and m o r p h o l o g i c a l o b s e r v a t i o n s  were f i x e d f o r 24 hours i n  b u f f e r e d f o r m o l - s a l i n e w h i l e those worms used f o r enzyme s t u d i e s were f i x e d f o r 24 hours a t 4°C i n acetone. Carnoy's fluid', pyridinium  formol-alcohol,  In a d d i t i o n ,  formol-calcium  and c e t y l  c h l o r i d e / f o r m a l i n were employed when r e q u i r e d f o r  s p e c i f i c techniques and f o r comparative purposes.  Specifically,  m a t e r i a l f i x e d i n Carnoy's f l u i d was r e q u i r e d f o r the B i e b r i c h s c a r l e t method (see page 30) and f o r m o l - c a l c i u m  f i x a t i o n was  used w i t h the Sudan b l a c k B method f o r bound l i p i d s .  Material  f i x e d i n Carnoy's f l u i d o r i n the f o r m a l i n - c o n t a i n i n g  fixatives  was dehydrated i n a l c o h o l , c l e a r e d i n xylene and embedded i n p a r a f f i n a t 52-59°C.  M a t e r i a l f i x e d i n c o l d acetone was  dehydrated i n acetone a t room temperature, c l e a r e d i n benzene and at  embedded i n p a r a f f i n a t 52°C. 5-10 jpi.  Formalin-fixed  P a r a f f i n s e c t i o n s were c u t  f r o z e n s e c t i o n s were a l s o used f o r  some o f the l i p i d t e s t s .  D.  Preparation  f o r Morphological  Studies.  For h i s t o l o g i c a l examination, s e c t i o n s were s t a i n e d w i t h one o f the f o l l o w i n g :  H a r r i s ' s alum hematoxylin,  Mallory's  t r i p l e s t a i n , Heidenhein's hematoxylin, E h r l i c h ' s hematoxylin or Weigertls hematoxylin, a c c o r d i n g Culling  (1963) and Ford  (1958).  to methods d e s c r i b e d by  6  E.  Preparation for Histochemical  The h i s t o c h e m i c a l techniques  Studies.  u t i l i z e d are  listed  below a c c o r d i n g to the m a t e r i a l to be demonstrated, i . e . , (1) carbohydrates, and  (5) l i p i d s .  procedures  (1)  (2) n u c l e i c a c i d s ,  Where no r e f e r e n c e s are given,  as d e s c r i b e d i n Pearse  Tests f o r  (3) enzymes,  (4) p r o t e i n s  standard  (1961) have been f o l l o w e d .  carbohydrates:  a.  Periodic acid Schiff  b.  PAS  (PAS)  (Culling,  a f t e r d i g e s t i o n w i t h 0.1%  1963).  malt d i a s t a s e f o r  30 minutes a t 37°C. c.  PAS  a f t e r a c e t y l a t i o n (and s a p o n i f i c a t i o n ) ( C u l l i n g ,  1963). d.  Combined a l c i a n blue-PAS method  (AB-PAS)  (Culling,  1963). e.  AB-PAS a f t e r a c i d h y d r o l y s i s to remove s i a l i c a c i d (Culling,  1963).  f.  0.3%  AB  i n 3% a c e t i c  g.  0.3%  AB  a f t e r d i g e s t i o n w i t h 0.05%  hyaluronidase  i n 0.1M  acid. testicular  phosphate b u f f e r (pH 5.5)  at  37°C f o r 2 hours. h.  Toluidine blue (0.1%-  (TB). . Various s t a i n  .005%), pH's  concentrations  (4.5 - 7) and s o l v e n t s  (30%  e t h a n o l , a c e t a t e b u f f e r or d i s t i l l e d water) were employed. i.  0.1%  aqueous TB a f t e r d i g e s t i o n w i t h  testicular  7  hyaluronidase. j.  0.1% azure A  (AA) i n 30% e t h a n o l  (Barka and Anderson,  1963). k.  0.2% AA  (pH 1.5,  pH 2.5,  pH.4.0) (Szirmai,  1.  Best's carmine.  m.  Methylene b l u e e x t i n c t i o n  n.  Aldehyde f u c h s i n - a l c i a n b l u e  1963).  (Barka and Anderson,  1963).  (AF-AB) (Spicer and  Meyer, 1960). o.  AF-AB a f t e r m e t h y l a t i o n  (and s a p o n i f i c a t i o n )  (Culling,  1963). p.  (2)  AB-AF  (Spicer and Meyer, 1960).  Tests f o r n u c l e i c  acids:  a.  Methyl green-pyronin Y (MGP)  b.  MGP  (Culling,  1963).  a f t e r d i g e s t i o n with 0.1%.pancreatic ribonuclease  a t 3 7°C f o r 1 hour. c.  Toluidine blue  (TB) a f t e r d i g e s t i o n w i t h p a n c r e a t i c  ribonuclease. d.  Feulgen method f o r DNA.  e.  MGP  o r TB a f t e r e x t r a c t i o n w i t h 4%  a c i d a t 90°C f o r 15 minutes  (3)  trichloroacetic  (Culling,  1963).  T e s t s f o r enzymes: As a c o n t r o l f o r a l l the enzyme t e s t s ,  s e c t i o n s were  i n c u b a t e d i n a medium i n which d i s t i l l e d water had been s u b s t i t u t e d f o r the s u b s t r a t e .  8  a.  A z o - c o u p l i n g technique f o r a l k a l i n e phosphatase w i t h the diazonium s a l t s F a s t b l u e RR and Red v i o l e t (Culling,  b.  Gomori's  1963). l e a d s u l p h i d e technique f o r a l k a l i n e  phosphatase c.  (Culling,  1963).  Naphthol AS-BI phosphate method f o r a c i d phosphatase w i t h the diazonium s a l t F a s t r e d .  d.  Gomori's (Culling,  e.  1963).  A z o - c o u p l i n g technique f o r n o n - s p e c i f i c e s t e r a s e (Culling,  (4)  l e a d s u l p h i d e technique f o r a c i d phosphatase  1963).  Tests f o r p r o t e i n s : a.  Biebrich s c a r l e t for basic proteins  (Spicer,  b.  Aqueous bromphenol b l u e f o r b a s i c p r o t e i n s  19 62).  (Johri  and Smyth, 195 6). c.  M e r c u r i c bromphenol  blue f o r general proteins  (MBPB) (Mazia e t a l . . d.  Mercury orange  (RSR) r e a c t i o n f o r -SH groups.  c o n t r o l technique was  (5)  1953).  not used w i t h t h i s  A  reaction.  Tests f o r l i p i d s : a.  L i l l i e and Ashburn's i s o p r o p a n o l o i l r e d 0 method (Culling,  b.  1963).  McManus's Sudan b l a c k B method lipids i n paraffin  sections.  (SBB) f o r compound  9  c.  Sudan b l a c k sections.  B method f o r n e u t r a l l i p i d s i n f r o z e n  10  RESULTS OF LIGHT MICROSCOPY  A. (1)  Morphological  Observations.  Fixation . Formol-saline,  t a i n i n g 0.5%  formol-calcium  10% f o r m a l i n con-  c e t y l p y r i d i n i u m c h l o r i d e (CPC/F) were found to  be the most s a t i s f a c t o r y f i x a t i v e s f o r observations  and  - but,  formol-calcium  morphological  f i x a t i o n has c e r t a i n  limitations for histochemical studies  (see C u l l i n g ,  1963;  p.  37).  CPC/F f i x a t i o n gave the b e s t p r e s e r v a t i o n of mucins and would be  the f i x a t i v e o f c h o i c e f o r f u t u r e i n v e s t i g a t i o n s o f  mucopolysaccharides. P l e r o c e r c o i d s f i x e d i n Carnoy's f l u i d to  l e s s than 1/2  contracted  of t h e i r o r i g i n a l length with a r e s u l t a n t  d i s t o r t i o n o f c u t i c u l a r s t r u c t u r e and,  i n most i n s t a n c e s , a  s e p a r a t i o n o f the c u t i c l e and s u b c u t i c u l a r l a y e r s . a l c o h o l f i x a t i o n a l s o produced some c u t i c u l a r although  not as much as d i d Carnoy's f l u i d .  Formol-  distortion Material fixed in  f o r m o l - a l c b h o l d i d appear to be more s e n s i t i v e to  ribonuclease  d i g e s t i o n than d i d t h a t f i x e d i n other formalin-based Acetone-fixed  material also exhibited considerable  o f the c u t i c l e and  fixatives.  separation  s u b c u t i c u l a r s t r u c t u r e s (see F i g u r e  20).  In some i n s t a n c e s t h i s made the exact l o c a l i z a t i o n o f enzyme sites  difficult.  11  (2)  Morphology o f the D i f f e r e n t i a t i n g P l e r o c e r c o i d . L i g h t microscope examination o f £>. s o l i d u s  p l e r o c e r c o i d s has demonstrated a p r o g r e s s i v e  change i n  c u t i c u l a r morphology c o r r e l a t e d w i t h an i n c r e a s e The  i n size.  s m a l l e s t forms s t u d i e d (L-5 mg) showed no obvious  segmentation and a v e r y t h i n ( < 4 p.) c u t i c l e w i t h no marked v a r i a t i o n i n t h i c k n e s s was present  (Figure 1 ) . A s u r f a c e  l a y e r which s t a i n e d w i t h most m o r p h o l o g i c a l s t a i n s l e s s i n t e n s e l y than the b a s a l c u t i c u l a r r e g i o n and an obvious m i c r o t h r i x border c o u l d be d i s c e r n e d .  The c e l l s o f the  s u b c u t i c u l a r parenchyma were s p i n d l e shaped and o r i e n t e d a t r i g h t anles  t o the c u t i c u l a r s u r f a c e .  some o f these c e l l s  (probably,  Long p r o j e c t i o n s from  i n these s m a l l worms,  muscle f i b r e s ) appeared to c r o s s the e n t i r e worm.  developing  Very t h i n  l a y e r s o f c i r c u l a r and l o n g i t u d i n a l muscle f i b r e s were observed immediately below the s u b c u t i c u l a r membrane.  A layer of  l o n g i t u d i n a l muscles, which would i n o l d e r worms separate the s u b c u t i c u l a r and medullary parenchyma, c o u l d a l s o be seen. The  s u b c u t i c u l a r membrane was v e r y t h i n and d i f f i c u l t t o  observe i n these s m a l l worms so t h a t u l t r a s t r u c t u r a l observations  were necessary t o c o n f i r m  i t s existence.  The  parenchymal n u c l e i were q u i t e l a r g e and s p h e r i c a l and contained  a single nucleolus.  Only a few c a l c a r e o u s  were observed s c a t t e r e d throughout the parenchyma. these m a g n i f i c a t i o n s ,  the cytoplasmic  corpuscles While, a t  tubules which connect the  parenchymal and c u t i c u l a r p o r t i o n s o f the tegument were v i s i b l e ,  12  t h e i r v e r y s m a l l s i z e made i t i m p o s s i b l e specific  to a s s i g n them t o  cells. P l e r o c e r c o i d s o f 10 mg gave evidence  o f segmentation  and a somewhat t h i c k e r c u t i c l e , b u t one which was s t i l l o f uniform  thickness.  By 20-30 mg the c h a r a c t e r i s t i c  pattern  of the d i f f e r e n t i a t e d p l e r o c e r c o i d c u t i c l e had developed. The  s u p e r f i c i a l p o r t i o n o f the c u t i c l e now was s i g n i f i c a n t l y  t h i c k e r than the velum and a m i c r o t h r i x border o f g r e a t e r d e n s i t y and depth was p r e s e n t . or  " c u t i c u l a r processes"  p o r t i o n o f the c u t i c l e .  Small c u t i c u l a r  protrusions  c o u l d be observed on the s u p e r f i c i a l I n t e r n a l l y , s e r i a l l y repeated g e n i t a l  rudiments c o u l d be seen. Pore c a n a l s became v i s i b l e w i t h  the l i g h t microscope  o n l y i n worms between 10 and 20 mg, although s t u d i e s have p r o v i d e d evidence as s m a l l as 1 mg.  f o r t h e i r e x i s t e n c e i n worms  In l a r g e r stages,  as a tube which p e n e t r a t e d the s u b c u t i c u l a r membrane.  ultrastructural  the pore c a n a l s appeared  the c u t i c l e and ended j u s t above The base o f the pore c a n a l was  formed by an e x t e n s i o n o f a c e l l  l y i n g i n the s u b c u t i c u l a r  parenchyma w i t h as many as s i x pore c a n a l s r e c e i v i n g processes  from a s i n g l e parenchymal c e l l .  The e l e c t r o n  microscope a l s o r e v e a l e d the presence o f d r o p l e t s , p o s s i b l y of a s e c r e t o r y nature,  i n the b a s a l r e g i o n o f the pore canals  (Figure 3 1 ) . The  fundamental p a t t e r n seen i n the 10-20 mg  p l e r o c e r c o i d was preserved  throughout f u r t h e r growth and  development.  A p l e r o c e r c o i d o f more than 200  mg had  a  s u p e r f i c i a l c u t i c u l a r t h i c k n e s s of 30-40 p. but the velum showed l i t t l e  i n c r e a s e d u r i n g development so t h a t a t a weight  of 200-250 mg  i t was  o n l y 3-4 ja t h i c k .  The  phenomenon o f  s p a t i a l l y l o c a l i z e d growth o f the c u t i c u l a r matrix may  be  d i r e c t l y r e l a t e d to the g r e a t e r number o f tegumental c e l l s connected to the s u p e r f i c i a l c u t i c l e . o f the c u t i c l e had  few  The  t h i n v e l a r region  tegumental c e l l s u n d e r l y i n g i t and  these c e l l s were p o s i t i o n e d p a r a l l e l to the velum s u r f a c e (Figure 7). The m i c r o t h r i x border i n c r e a s e d i n depth d u r i n g development, r e a c h i n g a t l e a s t 25 p., as was  particularly  e v i d e n t i n s e c t i o n s through the extreme a n t e r i o r and p o s t e r i o r segments.  The  c u t i c u l a r processes  also increased  i n s i z e w i t h p l e r o c e r c o i d growth so t h a t i n v e r y l a r g e worms they a t t a i n e d a h e i g h t of 2-4 JJL. and  The  pore c a n a l s were l a r g e r  i n g r e a t e r number per segment i n worms o f l a r g e r s i z e .  In many cases (Figure 2 ) .  they took on a f l a s k - l i k e or h o u r g l a s s  In some i n s t a n c e s the base of the pore c a n a l  became enlarged, without,  shape  b u l g i n g a g a i n s t the s u b c u t i c u l a r membrane  however, p e n e t r a t i n g i t .  Although no q u a n t i t a t i v e  data have been c o l l e c t e d on t h i s p o i n t , i t d i d appear t h a t pore c a n a l s were more numerous i n the a n t e r i o r p a r t of superficial cuticle.  the  C e r t a i n l y , l a r g e numbers of pore c a n a l s  were f r e q u e n t l y observed i n t h i s r e g i o n i n s t a n c e s grouped together  (Figure 7), i n many  i n a s m a l l area of the  cuticle.  14  No attempt has y e t been made t o determine whether a c o n s t a n t r a t i o e x i s t s throughout  growth between the number o f pore  c a n a l s and the area o f the c u t i c u l a r s u r f a c e .  Although  c u t i c u l a r processes have never been observed i n the velum, pore c a n a l s were seen i n t h i s r e g i o n w i t h the l i g h t  microscope  i n p l e r o c e r c o i d s o f more than 200 mg.  B. (1)  Histochemical Results.  D i s t r i b u t i o n o f Carbohydrates. The  PAS r e a c t i o n i s based on the f a c t t h a t c e r t a i n  t i s s u e elements  y i e l d aldehydes  o x i d i z a t i o n by p e r i o d i c a c i d . demonstrated  as r e a c t i o n products  after  These aldehydes may then be  w i t h a S c h i f f reagent.  According to Hotchkiss  (1948) any substance w i t h the f o l l o w i n g p r o p e r t i e s w i l l g i v e a p o s i t i v e r e s u l t w i t h the PAS method: a.  .The substance must c o n t a i n the 1:2 g l y c o l grouping, or the e q u i v a l e n t amino o r  .  alkyl-amino d e r i v a t i v e , o r the o x i d a t i o n product CHOH-CO.  b.  I t must not d i f f u s e away i n the course o f fixation.  c.  I t must give an o x i d a t i o n product which i s not  d.  diffusible.  S u f f i c i e n t c o n c e n t r a t i o n must be p r e s e n t t o give a detectable f i n a l c o l o u r .  15  With the use  o f c o n t r o l s and  supplementary s t a i n i n g techniques  i t i s p o s s i b l e to d i s t i n g u i s h between the wide v a r i e t y o f substances demonstrated by  t h i s method.  In a l l the worms t e s t e d , m a t e r i a l t h a t was p o s i t i v e and  diastase  l a b i l e was  assumed to be  PAS  glycogen.  The  r e s u l t s of the Best's carmine method were i d e n t i c a l to those o b t a i n e d w i t h the PAS  r e a c t i o n , thus p r o v i d i n g  of t h i s i n t e r p r e t a t i o n . quantities  Glycogen was  confirmation  found i n v a r y i n g  (based on r e l a t i v e i n t e n s i t i e s o f r e a c t i o n )  p a r t s of p l e r o c e r c o i d s except i n the c u t i c l e and membrane. deposited  in a l l  subcuticular  Q u a n t i t i e s of glycogen, i n muscle t i s s u e , were among the muscle f i b e r s .  concentrations  In worms o f 7-20  of glycogen were d e t e c t e d  mg,  dense  i n the c o r t i c a l  and  m e d u l l a r y parenchyma; the d e n s i t y g e n e r a l l y b e i n g  dependent  on the c e l l u l a r mass of any  also  region.  i n the s u b c u t i c u l a r parenchyma and w i t h the g r e a t e s t c o n c e n t r a t i o n s tegumental c e l l s .  Glycogen was  present  s u b c u t i c u l a r muscle l a y e r s  i n the r e g i o n of  the  Although the glycogen c o n c e n t r a t i o n s  of  the c o r t i c a l and m e d u l l a r y parenchyma remained r e l a t i v e l y constant  throughout growth, l i t t l e  glycogen c o u l d  be  demonstrated i n the tegumental c e l l s i n worms o f more than 20 mg.  But,  l a r g e q u a n t i t i e s o f glycogen were  i n the r e s t o f the s u b c u t i c u l a r parenchyma. r e a c t i o n i n t e n s i t y no marked g r a d i e n t s c o u l d be  detected  concentrated  On the b a s i s  i n glycogen  of  concentration  over the a n t e r i o r - p o s t e r i o r a x i s .  A glycogen e x t r a c t i o n from whole p l e r o c e r c o i d s  (KOH  16  digestion, ethanol  p r e c i p i t a t i o n method) a l s o r e v e a l e d the  presence o f l a r g e q u a n t i t i e s o f t h i s substance.  A sample  of 15 p l e r o c e r c o i d s o f a l l stages o f development was found to c o n t a i n 16.1% glycogen  (wet w e i g h t ) .  This f i g u r e i s  i d e n t i c a l to t h a t o f £>. s o l i d u s p u b l i s h e d b y Hopkins Following  (1950) .  d i a s t a s e d i g e s t i o n a marked PAS response  was r e t a i n e d i n two r e g i o n s o f the c u t i c l e ,  the s u b c u t i c u l a r  membrane and the b a s a l o r g a n e l l e s o f the pore c a n a l s . a d d i t i o n , most stages  In  examined had an e x t r a c u t i c u l a r coat o f  PAS p o s i t i v e m a t e r i a l which was c o n c e n t r a t e d  i n the r e g i o n  of the m i c r o t h r i x border, c l e a r l y e x t e r n a l to the c u t i c u l a r surface.  The PAS r e a c t i v i t y o f the c u t i c u l a r matrix  c o n s i d e r a b l y from almost e n t i r e l y negative  varied  i n s m a l l and  medium s i z e d (7-150 mg) p l e r o c e r c o i d s t o v e r y p o s i t i v e i n large  (> 200 mg) forms.  In worms between 7 and 80 mg the b u l k  of the c u t i c u l a r matrix was PAS negative positive.  or only  faintly  A l l , however, had a c l e a r l y p o s i t i v e l a y e r o f PAS  p o s i t i v e m a t e r i a l about 1% fx below the c u t i c l e s u r f a c e .  This  l a y e r was most s h a r p l y d e l i n e a t e d e x t e r n a l l y , b u t i n t e r n a l l y the r e a c t i v i t y decreased over a d i s t a n c e o f 1-2 ja u n t i l i t matched t h a t o f the g e n e r a l m a t r i x .  In l a r g e r worms,  i . e . 90-200 mg, the PAS p o s i t i v e l a y e r appeared a t t h e c u t i c u l a r s u r f a c e i n the s u p e r f i c i a l r e g i o n and i n c l u d e d the c u t i c u l a r processes.  The PAS r e a c t i v i t y r a p i d l y decreased  i n t e r n a l l y beyond the f i r s t 2-3 ja o f the c u t i c u l a r s u r f a c e . There were i n d i c a t i o n s t h a t the PAS p o s i t i v e l a y e r remained below the c u t i c u l a r s u r f a c e i n the velum and i n the extreme  17  p o s t e r i o r r e g i o n o f the s u p e r f i c i a l c u t i c l e i n worms over 90 mg.  The r e a c t i v i t y o f the c u t i c u l a r matrix  with s i z e .  also increased  The c u t i c l e o f a 138 mg p l e r o c e r c o i d was PAS  p o s i t i v e o n l y a t the s u r f a c e b u t worms o f 204 and 263 mg showed an i n t e n s e g e n e r a l c u t i c u l a r response t o the PAS s t a i n . The 263 mg worm was i n t e n s e l y p o s i t i v e i n i t s response so t h a t l i t t l e o r no d i f f e r e n c e i n r e a c t i v i t y between the s u r f a c e l a y e r and the g e n e r a l c u t i c u l a r matrix was observed. The PAS p o s i t i v e response o f the s u b c u t i c u l a r membrane was d e f i n i t e and c o n s t a n t  i n a l l stages  (Figure 3 ) .  A marked PAS p o s i t i v e response was g i v e n by the b a s a l o r g a n e l l e s o f the pore c a n a l s .  This response was p a r t i c u l a r l y obvious  where the c u t i c u l a r matrix was PAS negative b u t a p o s i t i v e response was g i v e n by the c u t i c l e s u r f a c e surrounding the e x t e r n a l p o r t i o n s o f the c a n a l s .  In the s m a l l e r worms the  tegumental c e l l s were o n l y f a i n t l y PAS p o s i t i v e b u t t h i s r e a c t i v i t y i n c r e a s e d w i t h s i z e o f worm and a r e l a t i v e l y i n t e n s e PAS p o s i t i v e response was g i v e n by the tegumental cells  i n worms o f more than 200 mg. Since substances other than carbohydrates  a p o s i t i v e PAS r e a c t i o n , the source o f c u t i c u l a r was c o n t r o l l e d by a c e t y l a t i o n . g l y c o l groups  (carbohydrates)  may g i v e  PAS r e a c t i v i t y  The PAS r e a c t i v i t y o f 1-2  i s removed by treatment w i t h  a mixture o f a c e t i c anhydride and p y r i d i n e ( a c e t y l a t i o n ) . Substances t h a t r e t a i n t h e i r PAS r e a c t i v i t y a f t e r a c e t y l a t i o n are assumed to be o f l i p i d c h a r a c t e r  (Barka and Anderson, 1963).  18  The  r e s t o r a t i o n o f h y d r o x y l groups and hence the r e s t i t i o n  of PAS s t a i n i n g may be accomplished by treatment w i t h 0.1 N KOH f o r 45 minutes a t room temperature. A f t e r 8 hours a c e t y l a t i o n , removed from a l l p l e r o c e r c o i d corpuscles of formol-saline  PAS r e a c t i v i t y was t o t a l l y  t i s s u e s except the c a l c a r e o u s  f i x e d worms.  A total restitution  of PAS r e a c t i v i t y r e s u l t e d from s a p o n i f i c a t i o n f o r 45 minutes a t room temperature.  These r e s u l t s confirmed the carbohydrate  nature o f a l l c u t i c u l a r and parenchymal substances, except the calcareous corpuscles,  demonstrated by the PAS r e a c t i o n .  A l c i a n blue i s a polybasic  copper p h t h a l o c y a n i n  d e r i v a t i v e which i n t e r a c t s w i t h c a r b o x y l and s u l f a t e groups both i n v i t r o and i n t i s s u e s e c t i o n s 1965). it  ( Q u i n t a r e l l i and D e l l o v o ,  When used i n c o n j u n c t i o n w i t h h y a l u r o n i d a s e  digestion  i s one o f s e v e r a l u s e f u l h i s t o c h e m i c a l methods f o r the  i d e n t i f i c a t i o n o f a c i d mucopolysaccharides.  Testicular  h y a l u r o n i d a s e s p e c i f i c a l l y removes h y a l u r o n i c chondroitin  a c i d and  sulphate A and C b u t does not h y d r o l y s e  chondroitin  s u l p h a t e B (see Curran, 1964; p. 195). The plerocercoids  b u l k o f the c u t i c u l a r matrix o f _S.  solidus  was u n r e a c t i v e w i t h A l c i a n b l u e although a  f a i n t response appeared i n worms o f more than 200 mg. was,  There  however, an i n t e n s e l y r e a c t i v e a l c i a n o p h i l i c l a y e r  discernable  i n worms o f from 10 to 118 mg i n the same  s u p e r f i c i a l l o c a t i o n as the PAS p o s i t i v e l a y e r above.  described  In the 118 mg worms the A l c i a n b l u e r e a c t i o n was most  19  i n t e n s e i n a narrow l a y e r under the c u t i c u l a r s u r f a c e  but i t  appeared t o be continuous w i t h the A l c i a n b l u e p o s i t i v e s u p e r f i c i a l to t h i s layer. the  In a l l worms o f more than 118 mg  p o s i t i v e A l c i a n b l u e response i n c l u d e d  surface  to a depth o f about 2 p..  c u t i c u l a r surface hyaluronidase.  region  o n l y the c u t i c u l a r  The r e a c t i v i t y o f the  was not a f f e c t e d by i n c u b a t i o n  i ntesticular  The tegumental c e l l s o f a l l s i z e s o f worms  gave a l i g h t p o s i t i v e response and the c a l c a r e o u s c o r p u s c l e s were i n t e n s e l y r e a c t i v e . organelles  Both the lumena and the b a s a l  o f the pore c a n a l s were u n r e a c t i v e w i t h A l c i a n b l u e . The  areas o f the c u t i c l e which r e a c t e d w i t h A l c i a n  b l u e c o u l d be determined w i t h g r e a t e r e x a c t i t u d e when t h i s s t a i n was combined w i t h the PAS method o r w i t h aldehyde fuchsin.  When these methods were employed i t became apparent  t h a t t h e r e was a l a y e r immediately beneath the c u t i c u l a r surface  which s t a i n e d  s e l e c t i v e l y with A l c i a n blue.  In small,  undifferentiated  forms t h i s l a y e r composed about 1/2 o f the  c u t i c u l a r matrix  (Figure 4 ) .  The c u t i c u l a r s u r f a c e ,  i n the  s m a l l forms, was i n t e n s e l y p o s i t i v e w i t h both the PAS and aldehyde f u c h s i n methods w h i l e the b a s a l lightly positive.  r e g i o n was o n l y  In l a r g e r worms the bulk o f the c u t i c u l a r  matrix was u n r e a c t i v e w i t h A l c i a n b l u e though i t d i d g i v e a l i g h t p o s i t i v e r e a c t i o n w i t h both PAS and aldehyde  fuchsin.  There was, however, an A l c i a n b l u e p o s i t i v e l a y e r  immediately  beneath the c u t i c u l a r s u r f a c e  In worms o f  100  (Figures  5 and 6).  mg the A l c i a n blue p o s i t i v e r e g i o n  a l s o extended i n t o  20  the b u l k o f t h e c u t i c u l a r m a t r i x i n t h e a n t e r i o r p o r t i o n o f t h e superficial region  (Figure 3 ) . A l a y e r o f apparently e x t r a -  c u t i c u l a r A l c i a n b l u e p o s i t i v e m u c i n s c o u l d a l s o be  observed  c o v e r i n g t h e velum and t h e extreme p o s t e r i o r t i p o f t h e superficial cuticle  (see F i g u r e 1 2 ) .  I t i s interesting to  n o t e t h a t a c i d h y d r o l y s i s , t o remove s i a l i c  acid,  caused a  s i g n i f i c a n t dimunition i n A l c i a n blue r e a c t i v i t y of the matrix i n two o u t o f t h r e e worms e x a m i n e d . The  m e t h y l a t i o n p r o c e d u r e was e m p l o y e d t o b l o c k  the s t a i n i n g o f both  sulphated mucopolysaccharides  d e s u l p h a t i n g them) a n d n o n - s u l p h a t e d  acid  (by  mucopolysaccharides  (by e s t e r i f y i n g t h e c a r b o x y l g r o u p s ) .  After methylation  the r e a c t i v i t y o f the c u t i c u l a r m a t r i x  to the aldehyde f u c h s i n -  A l c i a i b l u e m e t h o d was t o t a l l y r e m o v e d .  However, t h e c u t i c u l a r  s u r f a c e remained a t l e a s t p a r t i a l l y r e a c t i v e w i t h fuchsin after methylation.  This r e s i s t a n c e t o methylation  i s c h a r a c t e r i s t i c of certain sulphated acid (Spicer,  aldehyde  mucopolysaccharides  19 6 0 ) . S u b s e q u e n t s a p o n i f i c a t i o n endowed t h e c u t i c l e w i t h an  even g r e a t e r r e a c t i v i t y than e x i s t e d p r e v i o u s l y .  Both the  aldehyde f u c h s i n and A l c i a n b l u e s t a i n s r e a c t e d i n t e n s e l y w i t h the  cuticle. T o l u i d i n e b l u e and azure A produce a metachromatic  response i n the presence o f o r i e n t e d negative Although response,  many d i f f e r e n t s u b s t a n c e s the technique  surface  charges.  can give a metachromatic  i s considered diagnostic f o r acid  21  mucopolysaccharides when a p p l i e d to t i s s u e s e c t i o n s , and when the  metachromasy i s i n t e n s e and a l c o h o l r e s i s t a n t ,  i t may be  taken as f i r m evidence f o r the e x i s t e n c e o f s u l p h a t e d a c i d mucopolysaccharides  (Barka and Anderson,  Azure A w i l l ,  1963).  above pH 3, form a metachromatic  p r e c i p i t a t e w i t h a l l a c i d mucopolysaccharides,  including  h y a l u r o n i c a c i d , b u t i f the pH i s lowered below t h i s the  point  c a r b o x y l group d i s s o c i a t i o n i s decreased and thus  hyaluronic acid staining i s extinguished.  However, the  s u l p h a t e d a c i d mucopolysaccharides c o n t a i n both c a r b o x y l and s u l p h a t e groups and a t a pH o f 1-2 the sulphate group i s s t i l l d i s s o c i a t e d so the d i s a c c h a r i d e w i l l b i n d one dye molecule. Hence, azure A metachromasia  a t pH 1.5 may be regarded as  s p e c i f i c f o r s u l p h a t e d a c i d mucopolysaccharides. Although the above i n t e r p r e t a t i o n i s t h e o r e t i c a l l y sound, of  S z i r m a i (1963) has observed d i f f e r e n t i a l  metachromasia  c h o n d r o i t i n s u l p h a t e when s t a i n e d w i t h azure A a t low pH's.  He a t t r i b u t e s t h i s r e s u l t not to the presence o f a non-sulphated mucopolysaccharide b u t t o a c o m p e t i t i v e e f f e c t o f the c a t i o n i c groups o f p r o t e i n s , r e l a t i v e l y i n c r e a s i n g by l o w e r i n g the pH. The r e s u l t s o f the metachromatic  s t a i n i n g procedures  a p p l i e d to s e c t i o n s of v a r i o u s s i z e d worms v a r i e d c o n s i d e r a b l y w i t h the techniques used.  When s e c t i o n s s t a i n e d w i t h t o l u i d i n e  blue or . 1 % azure A i n 3 0 % e t h a n o l were examined i n d i s t i l l e d water,  the e n t i r e c u t i c u l a r matrix gave a b r i l l i a n t  response.  metachromatic  A f t e r s e c t i o n s were a i r - d r i e d and c l e a r e d i n x y l o l  22  t h i s metachromatic response was  reduced to the &  type.  However, the more s u p e r f i c i a l p o r t i o n o f the c u t i c l e  (not  i n c l u d i n g the s u r f a c e ) o f 24 and 44 mg worms became orthochromatic  a f t e r t h i s treatment w h i l e i n a 123 mg worm  the e n t i r e c u t i c l e ,  i n c l u d i n g the c u t i c u l a r  remained metachromatic  (/3)  (Figure 13).  same 12 3 mg worm were dehydrated  processes,  If sections of t h i s  i n a l c o h o l p r i o r to c l e a r i n g  and mounting, the s u p e r f i c i a l p o r t i o n of the c u t i c l e became orthochromatic w h i l e the r e s t o f the c u t i c l e gave a metachromatic response.  T h i s orthochromatic  p o r t i o n o f the c u t i c l e was  s t a i n i n g of the  observed,  a f t e r a l c o h o l dehydration,  i n s e c t i o n s o f 10 worms of from 10 to 150 mg. was  superficial  However, there  a t h i n l a y e r o f metachromasy a t the extreme c u t i c u l a r  s u r f a c e i n a l l i n s t a n c e s , and the b a s a l o r g a n e l l e s o f the pore c a n a l s and the s u b c u t i c u l a r membrane were orthochromatic (see F i g u r e 2 ) . was  Many s e c t i o n s a l s o possessed a l a y e r which  e x t e r n a l to the c u t i c u l a r processes and which gave a  metachromatic response.  I t i s probable t h a t the areas which  were orthochromatic w i t h t o l u i d i n e blue are the same as  those  which r e a c t e d most i n t e n s e l y w i t h a l c i a n b l u e . Sections s t a i n e d with t o l u i d i n e blue a f t e r hyaluronidase d i g e s t i o n showed s i g n i f i c a n t l y d i m i n i s h e d c u t i c u l a r metachromasia (Figures 13 and 14).  A f t e r t h i s treatment  some areas o f the  c u t i c l e became completely n o n r e a c t i v e and the p o s i t i v e  response  which remained l i k e l y r e s u l t e d from the presence of c u t i c u l a r RNA  (see page 26 and F i g u r e s 15 and  16).  23  When s e c t i o n s s t a i n e d w i t h a z u r e A a t pH 4.0 were examined i n d i s t i l l e d w a t e r , t h e e n t i r e d i s p l a y e d a "Y t h e /3  metachromasy.  T h i s r e s p o n s e was r e d u c e d t o  form a f t e r a i r d r y i n g and c l e a r i n g ,  alcohol dehydration remained.  cuticle  and f o l l o w i n g  o n l y a t h i n s u r f a c e l a y e r o f ft m e t a c h r o m a s y  S e c t i o n s s t a i n e d w i t h a z u r e A a t pH 1.5 showed no  r e a c t i v i t y o f the c u t i c u l a r matrix b u t d i d d i s p l a y a t h i n , i n t e n s e l y s t a i n i n g surface l a y e r which remained after alcohol dehydration  visible  (See F i g u r e s 9, 10 a n d 1 1 ) .  This  r e a c t i v i t y was l o c a t e d i n o n l y t h e m o s t s u p e r f i c i a l r e g i o n o f the c u t i c l e  and i n c l u d e d t h e s u r f a c e , b u t n o t t h e m a t r i x , o f  the c u t i c u l a r processes.  I n some i n s t a n c e s t h e s u p e r f i c i a l  r e a c t i v e l a y e r extended over  a s much a s 2/3 o f t h e l e n g t h  o f a pore c a n a l b u t t h e b a s a l p o r t i o n s o f pore c a n a l s were t o t a l l y unreactive i n a l l cases. C u t i c u l a r r e a c t i v i t y w i t h a z u r e A a t pH 1.5 was not diminished by i n c u b a t i o n i n t e s t i c u l a r In f a c t ,  hyaluronidase.  t h e r e a c t i v i t y o f t h e g e n e r a l c u t i c u l a r m a t r i x was  more i n t e n s e i n t h e h y a l u r o n i d a s e  t r e a t e d s e c t i o n s than i n  t h e c o n t r o l s i n 1/3 o f t h e s e c t i o n s o b s e r v e d .  I n those  i n s t a n c e s w h e r e a z u r o p h i l i a was i n c r e a s e d b y d i g e s t i o n , t h e r e a c t i v e areas were extended t o i n c l u d e a l l o f t h e c u t i c u l a r matrix except  t h e most s u p e r f i c i a l  the p r e v i o u s l y observed  1-2 p., i n a d d i t i o n t o  l a y e r on t h e c u t i c u l a r  surface.  The m e t h y l e n e b l u e e x t i n c t i o n p o i n t o f t h e c u t i c l e was f o u n d t o l i e b e t w e e n pH 3.6 a n d 4 . 1 .  A t pH 3.6 a f a i n t  24  c u t i c u l a r response c o u l d be a t pH 4.1  determined i n a water mount  the e n t i r e c u t i c l e s t a i n e d  b l u e e x t i n c t i o n p o i n t w i t h i n the t h a t which would be  intensely.  A methylene  above range approximates  expected from the presence of a  non-  s u l p h a t e d a c i d mucopolysaccharide such as h y a l u r o n i c A f t e r s e c t i o n s were c l e a r e d and mounted a f a i n t response c o u l d a l s o be 2.6  and  3.2  detected i n sections  which would be  and  acid.  surface  incubated a t  pH  expected i f sulphate groups were  present i n the macromolecules l o c a t e d a t the c u t i c u l a r  surface.  In view of the preceeding r e s u l t s i t would appear t h a t a t l e a s t three separate l a y e r s of a c i d mucins can d e t e c t e d i n the  cuticle:  a.  A surface  b.  A l a y e r immediately beneath the s u r f a c e reacts  sialic  the  b u l k of the c u t i c l e ,  and  be  contains carboxyl  composed of h y a l u r o n i c  D i s t r i b u t i o n of n u c l e i c  nuclease d i g e s t i o n was was  assumed to be a.  may  acid.  groups,  acid.  acids.  Methyl green-pyronin s t a i n i n g preceded by  RNA  which  A t h i r d l a y e r which, i n l a r g e r worms, composes  may  (2)  l a y e r of s u l p h a t e d a c i d mucopolysaccharides.  i n t e n s e l y w i t h a l c i a n b l u e and which  contain c.  be  ribo-  used f o r the i d e n t i f i c a t i o n o f present i n those s t r u c t u r e s  were p y r o n i n p o s i t i v e b e f o r e digestion,  RNA.  which:  ribonuclease  25  b.  d i d not s t a i n w i t h p y r o n i n a f t e r  ribonuclease  digestion. The  Feulgen t e s t was used f o r the i d e n t i f i c a t i o n o f DNA and  since  o n l y n u c l e i o f d e f i n i t e c e l l s were p o s i t i v e no f u r t h e r  c o n t r o l s were c a r r i e d o u t . observed i n any p o r t i o n  No Feulgen p o s i t i v e response was  o f the c u t i c l e .  Some p o r t i o n s o f the  c u t i c l e were p o s i t i v e f o r methyl green when methyl green and p y r o n i n s t a i n s were a p p l i e d  separately  (Davenport, 1960) b u t  t h i s response was not g i v e n by the combined methyl greenp y r o n i n method o f Jordan and Baker The  ( C u l l i n g , 1963).  b u l k o f the c u t i c u l a r matrix, the tegumental  c e l l s and the c e l l s o f the parenchyma were i n a l l worms s t u d i e d .  In a d d i t i o n ,  pyronin p o s i t i v e material surface.  two bands o f i n t e n s e l y  c o u l d be d e t e c t e d near the c u t i c u l a r  The f i r s t band was composed o f e x t r a c u t i c u l a r  mucin-like material microthrix worms.  pyronin-positive  located within  the bounds o f the  b o r d e r and was most commonly observed i n s m a l l e r  The second band o f p y r o n i n p o s i t i v e m a t e r i a l  was  immediately below the c u t i c u l a r s u r f a c e i n worms o f l e s s than 100 mg b u t i n c l u d e d  the c u t i c u l a r s u r f a c e and c u t i c u l a r  processes i n a l l worms l a r g e r than t h i s .  Although the p y r o n i n  p o s i t i v e response o f the c u t i c u l a r matrix, the tegumental c e l l s and  the parenchymal c e l l s was l a r g e l y removed by d i g e s t i o n  w i t h r i b o n u c l e a s e , the p o s i t i v e s t a i n i n g c h a r a c t e r i s t i c s o f the  s u p e r f i c i a l bands were not a f f e c t e d by t h i s enzyme  treatment  (Figures  17 and 18).  26  Toluidine  b l u e s t a i n i n g proved to be a more s e n s i t i v e  i n d i c a t o r o f RNA'ase a c t i v i t y than d i d the methyl green-pyronin method.  E x t r a c t i o n w i t h h o t t r i c h l o r o a c e t i c a c i d removed the  b u l k o f the c u t i c u l a r t o l u i d i n e b l u e metachromasia parenchymal metachromasia corpuscles.  Very l i t t l e  except t h a t o f the c a l c a r e o u s e f f e c t on p y r o n i n s t a i n i n g  observed when the e x t r a c t i o n was pyronin s t a i n .  and a l l  was  f o l l o w e d by the methyl green-  The e f f e c t o f RNA'ase treatment was  similar  to h o t t r i c h l o r o a c e t i c a c i d i n t h a t the t o l u i d i n e b l u e metachromasia  o f the c u t i c l e was  t o t a l l y removed (Figures  (3)  s i g n i f i c a n t l y reduced, but not  15 and 16).  D i s t r i b u t i o n of enzymes. Both a l k a l i n e and a c i d phosphatase appear to be  p r e s e n t i n the c u t i c l e o f a l l s i z e s o f worms. the  Incidentally,  a z o - c o u p l i n g techniques a l l o w e d a c c u r a t e l o c a l i z a t i o n o f  phosphatases b u t the Gomori methods were i n f e r i o r i n t h i s respect.  The e n t i r e c u t i c l e plus the tegumental c e l l s were  reactive  f o r a l k a l i n e phosphatase and i n the  sexually  mature forms the u t e r i n e w a l l s were r e a c t i v e  19).  The r e a c t i v i t y o f the pore c a n a l s was  variable  though the lumen was  f a s t blue technique.  the  anterior  (Figure  s l i g h t and  r e a c t i v e w i t h the  Although l i t t l e v a r i a t i o n i n a l k a l i n e  phosphatase r e a c t i v i t y e x i s t e d c u t i c l e there was  intensely  larger  through the depth o f the  an enhanced a c t i v i t y i n the velum and i n  portion  of the s u p e r f i c i a l c u t i c l e  (Figure  19).  27  The  tegumental c e l l s i n these same areas a l s o demonstrated  increased  activity. With the Gomori method f o r a c i d phosphatase  c u t i c u l a r r e a c t i o n was and  the more b a s a l  present i n the  confined  r e g i o n s and  to the most s u p e r f i c i a l a c i d phosphatase a c t i v i t y  lumen of the pore c a n a l s  l a t t e r r e a c t i o n s i t e c o u l d not be any  the  (Figure 20).  was  This  r e l a t e d to r e a c t i v i t y of  p a r t i c u l a r tegumental c e l l s and where p r e c i p i t a t e s were  observed i n the  tegumental r e g i o n  t h e i r appearance  t h a t they r e p r e s e n t e d d i f f u s i o n a r t e f a c t s r a t h e r enzyme s i t e s .  indicated  than a c t u a l  A p o s i t i v e response f o r a c i d phosphatase  was  a l s o g i v e n by  the  The  p o s i t i v e r e a c t i o n of calcareous corpuscles  a l l worms does not  t e s t e s o f s e x u a l l y mature forms. obtained i n  i n d i c a t e the presence of a c i d phosphatase  as t h i s r e a c t i o n was  a l s o g i v e n by c o n t r o l s e c t i o n s .  The  extreme v a r i a b i l i t y of the r e s u l t s o f the Gomori method f o r a c i d phosphatase made d i f f i c u l t the d e t e c t i o n o f a c t i v i t y over the l e n g t h there was  In some areas  so dense t h a t no meaningful l o c a l i z a t i o n  obtained.  D e s p i t e these handicaps i t c o u l d  observed t h a t the r e g i o n o f most r e p r o d u c i b l e included I t was  gradients  l i t t l e or no c u t i c u l a r r e a c t i o n w h i l e i n o t h e r s  the r e a c t i o n was would be  o f the c u t i c l e .  of any  the velum and  activity  the a n t e r i o r s u p e r f i c i a l c u t i c l e .  i n these same r e g i o n s t h a t the b a s a l  c u t i c u l a r matrix a l s o r e a c t e d The  be  p o r t i o n of  consistently.  naphthol AS-BI phosphate method f o r a c i d  the  28  phosphatase i n d i c a t e d a more widespread occurrence o f t h i s enzyme than d i d the Gomori method and  the  to mimic those given by the a z o - c o u p l i n g phosphatase. r e a c t i v i t y was  The  s i m i l a r i n each segment to t h a t  seen i n the velum and  region.  method f o r a l k a l i n e  d i s t r i b u t i o n of c u t i c u l a r a c i d phosphatase  f o r a l k a l i n e phosphatase s i n c e an i n t e n s e be  results.appeared  the e n t i r e c u t i c u l a r matrix and  o f the worms but  activity  the a n t e r i o r s u p e r f i c i a l  In a d d i t i o n , a l e s s i n t e n s e  s i t e s of esterase  described  r e a c t i o n was  could cuticle seen throughout  i n the tegumental c e l l s .  a c t i v i t y c o u l d be  detected  t h i s r e s u l t must be  i n any  regarded as  No  portion  tentative  pending the a p p l i c a t i o n of more s e n s i t i v e procedures.  (4)  D i s t r i b u t i o n of The  proteins.  mercuric brom-phenol b l u e method i s a  general  p r o t e i n s t a i n w h i l e the aqueous method i s supposedly s p e c i f i c for basic proteins  ( J o h r i and  Smyth, 1956).  brom-phenol b l u e method proved to be  The  non-specific  r e s u l t s were judged u n r e l i a b l e as a l l p o r t i o n s  of  p l e r o c e r c o i d were s t a i n e d w i t h equal i n t e n s i t y .  aqueous and  the  the The  intensity  o f c u t i c u l a r s t a i n i n g v a r i e d w i t h p l e r o c e r c o i d s i z e when the m e r c u r i c brom-phenol blue method was both the m i c r o t h r i x border and i n t e n s e l y w h i l e the pore c a n a l s and  applied.  the c u t i c u l a r matrix  stained  appeared as empty channels  the s u b c u t i c u l a r membrane gave a l i g h t but  As worm weight i n c r e a s e d  In s m a l l worms  there was  p o s i t i v e response.  a progressive  reduction  in  29  the response o f the c u t i c u l a r matrix so t h a t i n worms o f more than 150 mg o n l y a f a i n t p o s i t i v e response c o u l d be observed.  The c u t i c u l a r s u r f a c e was, however, i n t e n s e l y  p o s i t i v e i n a l l stages examined, a response which was p a r t i c u l a r l y obvious i n those l a r g e r stages i n which the general  m a t r i x responded weakly.  (See Figure  8.)  In the  l a r g e s t stage examined (263 mg) the r e a c t i o n extended inward from the c u t i c u l a r s u r f a c e  to a depth o f 1-2 ju and i n c l u d e d  the w a l l s o f the pore c a n a l s .  The most intense  though, was a t the c u t i c u l a r s u r f a c e . mucin-like  material  reaction,  Extra-cuticular  found i n the r e g i o n o f the m i c r o t h r i x  border a l s o gave a p o s i t i v e response to the mercuric bromphenol b l u e method ( i . e . ,  general  protein stain).  The  e n t i r e p l e r o c e r c o i d gave a l i g h t p o s i t i v e response to the Millon reaction  ( s p e c i f i c f o r t y r o s i n e ) b u t no l o c a l i z e d  s t a i n i n g c o u l d be  detected.  When the s t a i n i n g i s c a r r i e d o u t a t a pH o f 9-10 B i e b r i c h s c a r l e t i s s p e c i f i c f o r n u c l e o h i s t o n e s and histone-like basic proteins a l s o been presented  (Spicer, 19 62)  strongly basic, histone-type, containing  (Spicer, 1962).  protein  (mucihistone) i n s i t e s An attempt to i d e n t i f y  i n formalin-fixed plerocercoids with Biebrich  s c a r l e t a t pH 9.5 was u n s u c c e s s f u l . corpuscles  f o r the presence o f  a c i d mucopolysaccharides.  basic proteins  Evidence has  reacted  Only the c a l c a r e o u s  c o n s i s t e n t l y and although a p o r t i o n o f the  c u t i c u l e s t a i n e d l i g h t l y t h i s can not be regarded as s i g n i f i c a n t  30  in  the absence o f a n u c l e a r r e a c t i o n .  When t h e same  staining  m e t h o d was a p p l i e d t o C a r n o y - f i x e d m a t e r i a l a v i v i d  orange-red  r e s p o n s e was g i v e n b y t h e n u c l e i a n d b y the c u t i c u l a r  surface.  The  m o s t i n t e n s e n u c l e a r r e s p o n s e was g i v e n b y t h e t e s t e s a n d  ovaries.  T h i s r e s p o n s e was o f a d e n s e r e d n a t u r e  the e n t i r e n u c l e a r s t r u c t u r e . cell The  and i n c l u d e d  The r e s p o n s e o f t h e t e g u m e n t a l  n u c l e i was e q u a l l y v i v i d b u t o f a f i n e l y g r a n u l a r cuticular surface stained b r i l l i a n t l y  which covered  both  over  nature.  a narrow band  t h e velum and s u p e r f i c i a l c u t i c l e and  which extended completely  i n t o the pore c a n a l s .  A  positive  r e s p o n s e may a l s o h a v e b e e n g i v e n b y t h e b a s a l o r g a n e l l e o f the pore c a n a l b u t t h i s c o u l d n o t be d e t e r m i n e d w i t h c e r t a i n t y as a l m o s t a l l o f t h e c u t i c l e h a d s e p a r a t e d  from  the s u b c u t i c u l a r t i s s u e i n t h e G a r n o y - f i x e d m a t e r i a l .  The  c a l c a r e o u s c o r p u s c l e s were a l s o r e a c t i v e , p a r t i c u l a r l y i n t h e membrane s u r r o u n d i n g cell  t h e c o r p u s c l e and i n t h e c o r p u s c l e -  nucleus. A g e n e r a l p o s i t i v e response t o the Mercury orange  r e a c t i o n f o r p r o t e i n s u l p h y d r y l g r o u p s was g i v e n b y a l l tissues.  The o n l y i n d i c a t i o n o f l o c a l c o n c e n t r a t i o n s was  observed  i n t h e c u t i c u l a r s u r f a c e o f a 263 mg p l e r o c e r c o i d  but  t h i s was n o t r e g a r d e d  a s s i g n i f i c a n t a n d no f u r t h e r  c o n t r o l s were c a r r i e d o u t f o r t h i s r e a c t i o n .  (5)  Distribution of lipids. The  Sudan b l a c k B a n d o i l r e d 0 m e t h o d s w e r e a p p l i e d  31  to frozen sections i n order concentrations  t o r e v e a l t h e p r e s e n c e o f any  of neutral l i p i d s  i n the c u t i c l e . A l l  p l e r o c e r c o i d t i s s u e s responded t o these i n t e n s e b u t n o n - s p e c i f i c manner.  s t a i n s i n a moderately  The v i t e l l a r i a  d i d , however,  give a granular b r i l l i a n t r e d response w i t h the o i l r e d 0 method, i n d i c a t i n g t h e p r e s e n c e o f f r e e l i p i d s  i n these  organs. Where f o r m a l i n - f i x e d , p a r a f f i n - e m b e d d e d t i s s u e i s u s e d , p o s i t i v e r e s u l t s w i t h t h e Sudan b l a c k m e t h o d " f o r m o l - f i x e d " l i p o p r o t e i n whose l i p i d phosphatide or cerebroside  (Pearse,  signify  component i s u s u a l l y  1961).  The  cuticular  m a t r i x was p o s i t i v e w i t h t h i s m e t h o d , p a r t i c u l a r l y i n worms o f l e s s t h a n 90 mg  (see F i g u r e s  21 t o 2 3 ) .  The m o s t  i n t e n s e l y p o s i t i v e r e s u l t s were g i v e n by t h e c u t i c l e o f a 10 mg p l e r o c e r c o i d w h i l e  the c u t i c l e of p l e r o c e r c o i d s of  150 mg was o n l y f a i n t l y p o s i t i v e . f i x e d worms e x a m i n e d t h e c a l c a r e o u s  In a l l formol-saline c o r p u s c l e s were p o s i t i v e  (see F i g u r e 22) b u t a l l p a r e n c h y m a l t i s s u e s , t h e s u b c u t i c u l a r membrane a n d t h e p o r e c a n a l s w e r e n e g a t i v e  or only  faintly  positive. Although t o be l o c a l i z e d ,  the c u t i c u l a r  the p r e v i o u s l y described surface layer of  t h e c u t i c l e was c l e a r l y v i s i b l e Sudan b l a c k .  response d i d n o t appear  i n sections stained with  The v i s i b i l i t y o f t h e s u r f a c e  l a y e r appeared to  be more a r e s u l t o f d i f f e r e n t r e f r a c t i v e p r o p e r t i e s t h a n o f stain  affinity. I t s h o u l d be n o t e d t h a t f o r m o l - c a l c i u m  i s a preferred  32  fixative  f o r t h e Sudan b l a c k m e t h o d f o r b o u n d l i p i d s .  more v i v i d  staining  Much  d i d r e s u l t when t i s s u e was f i x e d i n  formol-calcium rather than i n formol-saline or formol-alcohol (see F i g u r e 2 4 ) .  33  RESULTS OF  (1)  ELECTRON MICROSCOPY  Morphology of d i f f e r e n t i a t e d As  plerocercoids.  d e s c r i b e d above (see  F i g u r e 2)  the  cuticular  matrix appears w i t h the  l i g h t microscope as a homogeneous  layer,  i n the  r e l a t i v e l y thick  s u p e r f i c i a l r e g i o n s and  t h i n n e r i n the velum, p e n e t r a t e d a t i n t e r v a l s by p o s s e s s i n g a s u r f a c e r e g i o n of p a p i l l a - l i k e p r o c e s s e s " and subcuticular  m i c r o t r i c h e s and  morphology (see  pore c a n a l s ,  "cuticular  terminated b a s a l l y by p  membrane which i s 2-3  Electron  much  a  thick.  microscopy r e v e a l e d a much more heterogeneous  F i g u r e 33).  The  superficial cuticle  was  thrown i n t o a s e r i e s of f o l d s or c u t i c u l a r processes which were continuous w i t h the 25  and  33).  The  general c u t i c u l a r matrix  c u t i c u l a r processes were t y p i c a l of a l l  p l e r o c e r c o i d s from 18-300 mg  but  none c o u l d be  p l e r o c e r c o i d s of l e s s than 10 mg c u t i c u l a r processes v a r i e d  (see  i n s i z e and  observed i n  F i g u r e 35).  The  i n h e i g h t from about 0.7  and were spaced a t i n t e r v a l s of 1-3 variation  (Figures  p.  to  Although c o n s i d e r a b l e  s p a c i n g of c u t i c u l a r processes  observed i n i n d i v i d u a l p l e r o c e r c o i d s ,  4^  the  was  processes from a  s p e c i f i c r e g i o n were s t r i k i n g l y uniform i n t h i s r e s p e c t . data have been c o l l e c t e d on v a r i a t i o n  in cuticular  but  the  i t would appear a t t h i s time t h a t  were found on c u t i c l e i n the  the  anterior  portion  r e g i o n overlapped by  of the the  largest  No  processes processes  superficial  velum  (i.e.,  the  same  34  r e g i o n demonstrating i n t e n s e enzyme a c t i v i t y ) . the bases o f the c u t i c u l a r processes the s t r u c t u r e s a f l a s k - l i k e shape o f the c u t i c u l a r processes  was  In some i n s t a n c e s  were c o n s t r i c t e d g i v i n g  (Figure 25).  continuous w i t h  The  t h a t of  general c u t i c l e and m i c r o t r i c h e s o r i g i n a t e d from adjacent  c u t i c u l a r surfaces.  process was  and  the  the  An unusual type o f c u t i c u l a r  observed on the p o s t e r i o r segments of a 94  plerocercoid. organized  matrix  These c u t i c u l a r processes  mg  were l o o s e l y  surrounded by q u a n t i t i e s of a mucous-like m a t e r i a l .  L i g h t microscope o b s e r v a t i o n s  have i n d i c a t e d t h a t l a r g e , l o o s e l y  organized  o f t h i s type may  c u t i c u l a r processes  of the more a n t e r i o r and p o s t e r i o r segments. processes  be  characteristic  No c u t i c u l a r  have been observed i n the v e l a r r e g i o n of  any  specimen. The velum and  c u t i c u l a r m i c r o t r i c h e s were present  the s u p e r f i c i a l regions of the c u t i c l e  i n both (Figure  the 25).  The velum m i c r o t r i c h e s were s h o r t e r and  appeared more r i g i d  than those of the s u p e r f i c i a l c u t i c l e .  The m i c r o t r i c h e s  of  _S. s o l i d u s d i f f e r e d i n some r e s p e c t from those of Hymenolepis diminuta and  (Rothman, 1963), D i p y l i d i u m caninum  Proteocephalus p o l l a n i c o l i  much longer,  (Threadgold,  (Threadgold, 1965).  r e a c h i n g lengths of 10 p. and more and  They were the e l e c t r o n  dense d i s t a l h a l f d i d not l i e a t an o b l i q u e angle to proximal 1965) .  p o r t i o n as d e s c r i b e d f o r J?. p o l l a n i c o l i  1962)  the  (Threadgold,  In £>. s o l i d u s the s u r f a c e plasma membrane covered  the e n t i r e m i c r o t h r i x  (Figures 2 7 to 34).  The b a s a l  region  35  c o n t a i n e d a dark core p o s s i b l y formed by a r i n g of twenty fibers  (see F i g u r e 26).  The d i s t a l p o r t i o n was  ovoid rather  than s p h e r i c a l or angular and had a c e n t r a l zone o f c l o s e l y packed f i b e r s surrounded by a zone o f e l e c t r o n l u c i d m a t e r i a l (see F i g u r e 27).  As y e t no p r o j e c t i o n s have been observed  p a s s i n g from the base o f a m i c r o t h r i x i n t o the c u t i c u l a r matrix. The c u t i c u l a r matrix was  composed of many c l o s e l y  packed v e s i c u l a r s t r u c t u r e s , each o f which was  bounded by  a double membrane.  general types:  These v e s i c l e s were of two  a l a r g e o v o i d type w i t h a membraneous e l e c t r o n l u c i d and a s m a l l e r r o d - l i k e type w i t h an e l e c t r o n dense Both types o f v e s i c l e may  be seen i n F i g u r e 28.  interior  interior.  In some areas  the v e s i c l e s were arranged i n s t a c k s w h i l e i n o t h e r areas they were randomly o r i e n t e d , but there was  no evidence o f  any r e g i o n a l c o n c e n t r a t i o n o f one type or the other w i t h i n the c u t i c u l a r matrix o f l a r g e p l e r o c e r c o i d s .  There  was,  however, a g r e a t e r c o n c e n t r a t i o n o f the r o d - l i k e v e s i c l e s i n the c u t i c u l a r processes than i n the c u t i c u l a r m a t r i x . I t would a l s o appear a t t h i s time t h a t the r o d - l i k e v e s i c l e s were c h a r a c t e r i s t i c o f s m a l l p l e r o c e r c o i d s and the o v o i d type appeared  l a t e r i n development.  L i t t l e c o u l d be  determined  about the m o r p h o l o g i c a l nature o f the c u t i c u l a r ground substance, but the c u t i c u l a r v e s i c l e s appeared  to occupy  more of the m a t r i x volume i n the l a r g e r p l e r o c e r c o i d s . evidence o f l a y e r i n g o f the c u t i c u l a r matrix c o u l d be  No seen  36  in  e l e c t r o n micrographs. Mitochondria  in  were g e n e r a l l y l o c a t e d i n t h e b a s a l  larger plerocercoids while  the m i t o c h o n d r i a larger  and  inclusions  had  i n e a r l y developing  were l o c a t e d t h r o u g h o u t t h e  a swollen  appearance.  included myelin  figures  p i n o c y t o t i c - l i k e v e s i c l e s and No  e v i d e n c e has  the h y p o t h e s i s  that  the  the  r e s u l t s obtained  caninum, b u t on  the  tegument o f _P.  f r o m the but  not  region  instances, like  29,  no  This  Threadgold's  the  30  31.  droplets  A  The  the  i s i n contrast  (1965)  sides of  o b s e r v e d was  pore c a n a l s Unlike  s o l i d u s pore  c u t i c l e was  parenchymal p o r t i o n o f the These t u b u l e s ,  compartments to  D.  observations  the  common f e a t u r e  (see F i g u r e  (Figure 30).  S.  support  D.  solidus  Microtriches  31),  arose  canals of  the  an  aggregation  and  i n some  contained caninum  membraneous s t r u c t u r e s c o u l d be  lumen o f  into  channels.  p o r e c a n a l s - o f _S.  and  region.  of pore c a n a l s  lumen o f  substance  1962), the  the  small,  pollanicoli.  terminal  electron lucid  were  (1962) i n a s t u d y o f  c u t i c u l a r s u r f a c e bounding the  terminal of  Threadgold  i n Figures  from the  29),  which would  epithelium.  u l t r a s t r u c t u r e of  illustrated  (see F i g u r e  i s subdivided  i n accordance with  The is  by  cuticle,  Other mature c u t i c u l a r  been obtained  i n t h e manner o f a t y p i c a l  forms  o c c a s i o n a l membraneous  cuticle  region  a mucous-  (Threadgold,  observed  crossing  canals.  connected,  at intervals,  tegument b y  w h i c h were v a c u o l a t e d  or  cytoplasmic solid,  to  the  tubules.  contained  37  numerous mitochondria  (Figure 3 2 ) . A b a s a l plasma membrane  c o u l d not be d e t e c t e d butthe b a s a l r e g i o n o f the c u t i c l e was penetrated,  to v a r y i n g depths, by numerous s m a l l channels which  were continuous w i t h the substance o f the s u b c u t i c u l a r membrane. Flame c e l l s which were commonly observed i n the parenchyma resembled those d e s c r i b e d f o r other helminths (Rothman, 1963; P a n t e l o u r i s and Threadgold, 19 63). "flame" contained the 9 + 2  about 80 c i l i a ,  Each  each o f which d i s p l a y e d  f i l a m e n t p a t t e r n t y p i c a l o f these s t r u c t u r e s . Although no attempt was made t o d i f f e r e n t i a t e  between parenchymal c e l l types, commonly observed.  c e r t a i n f e a t u r e s were  A t y p i c a l n u c l e a r s t r u c t u r e was seen i n  g l u t a r a l d e h y d e - f i x e d and u r a n y l a c e t a t e 37).  The chromatin was r e c o g n i s a b l e  stained tissue  as dense granular  l o c a t e d around the n u c l e a r p e r i p h e r y and a double membrane w i t h  pores was e v i d e n t .  (Figure material  nuclear  In some i n s t a n c e s ribosomes c o u l d  be seen on the n u c l e a r membrane and i n a t l e a s t one i n s t a n c e the outer n u c l e a r membrane appeared continuous w i t h reticulum.  Granular  the endoplasmic  endoplasmic r e t i c u l u m was found i n a l l  parenchymal c e l l s observed.  In F i g u r e 37 the p o r t i o n o f the  g r a n u l a r endoplasmic r e t i c u l u m l o c a t e d along had ribosomes o n l y on the i n t e r n a l membrane.  the plasma membrane Figures  38 and  39 i l l u s t r a t e a type o f endoplasmic r e t i c u l u m observed i n the parenchyma, o f the two p l e r o c e r c o i d s .  T h i s type o f endoplasmic  r e t i c u l u m i s a l s o found i n p r o t e i n - s e c r e t i n g g l a n d u l a r c e l l s o f vertebrates  (Fawcett, 1966).  38  (2)  Morphology o f u n d i f f e r e n t i a t e d p l e r o c e r c o i d s . Plerocercoids  o f l e s s t h a n 5 mg h a d a c u t i c u l a r  d e p t h o f a p p r o x i m a t e l y 3 p a n d a s u b c u t i c u l a r membrane a b o u t .2 p t h i c k .  The m i c r o t r i c h e s w e r e i d e n t i c a l t o t h o s e  seen i n l a r g e r p l e r o c e r c o i d s  and a t t a i n e d a l e n g t h o f a t l e a s t  10 p..  showed no i n d i c a t i o n o f c u t i c u l a r  The c u t i c u l a r s u r f a c e  processes.  Some m i c r o g r a p h s showed t h e p r e s e n c e o f many  small surface  invaginations,  resembling pinocytotic v e s i c l e s .  Large v e s i c l e s (about \ p i n diameter) c o u l d a l s o be seen i n the c u t i c u l a r m a t r i x ; cuticle  u s u a l l y i n t h e o u t e r m o s t 1/3 o f t h e  ( F i g u r e 3 5 ) . These v e s i c l e s d i d n o t have a s t r u c t u r e d  i n t e r i o r a n d t h e i r e l e c t r o n d e n s i t y was i d e n t i c a l w i t h the  e x t r a c u t i c u l a r area.  They w e r e , h o w e v e r e n c l o s e d  that of by a  d o u b l e membrane a n d a p p e a r e d t o b e o f m i t o c h o n d r i a l  origin  (see F i g u r e  scattered  evenly  34). Myelin  f i g u r e s c o u l d a l s o be seen  throughout the c u t i c u l a r matrix  i n much g r e a t e r  than observed i n any o f the l a r g e r p l e r o c e r c o i d s .  numbers  Structures  r e s e m b l i n g t h e m y e l i n f i g u r e s o f t h e c u t i c l e were o b s e r v e d i n the  cytoplasm o f the tegumental c e l l s and i n the cytoplasmic  tubules  connecting The  these c e l l s  to the c u t i c l e .  r o d - l i k e e l e c t r o n dense v e s i c l e s appeared t o  predominate i n the c u t i c l e o f small p l e r o c e r c o i d s , instances  to the almost t o t a l e x c l u s i o n of the l a r g e r  e l e c t r o n l u c i d type. it  i n some  Although these r e s u l t s are t e n t a t i v e  d i d appear t h a t t h e c u t i c l e o f very  small  plerocercoids  was c h a r a c t e r i z e d b y t h e p r e s e n c e o f t h e s e r o d - l i k e v e s i c l e s ,  39  with  the e l e c t r o n - l u c i d type appearing l a t e r i n development.  E x a m i n a t i o n o f t h e c u t i c l e o f a 22 mg p l e r o c e r c o i d the  revealed  p r e s e n c e o f l a r g e r numbers o f r o d - l i k e v e s i c l e s i n t h e  c u t i c u l a r processes than i n the a d j o i n i n g matrix.  A  greater  number o f t h e r o d - l i k e v e s i c l e s t h a n o f t h e e l e c t r o n - l u c i d t y p e was a l s o v i s i b l e previously,  i n t h e v e l u m o f t h e worm.  As  t h e c u t i c u l a r p r o c e s s e s o f much l a r g e r  a l s o seemed t o c o n t a i n  a high  proportion  described plerocercoids  of the r o d - l i k e  vesicles. Numerous m i t o c h o n d r i a w e r e o b s e r v e d t h r o u g h o u t t h e c u t i c l e of the small  plerocercoids  a n d a l t h o u g h some c o n c e n -  t r a t i o n a t t h e b a s e e x i s t e d i t was n o t a s m a r k e d a s i n l a r g e r forms.  I n a d d i t i o n , many l a r g e membraneous  r e s e m b l i n g m i t o c h o n d r i a were l o c a t e d i n the b a s a l c u t i c l e o f a 1.5 mg p l e r o c e r c o i d .  structures region  o f the  The e x a c t n a t u r e o f t h e s e  s t r u c t u r e s has n o t , however, been d e t e r m i n e d . No p o r e c a n a l s  have y e t been found i n p l e r o c e r c o i d s  o f l e s s t h a n 5 mg b u t s e v e r a l s t r u c t u r e s organelles  have been o b s e r v e d i n t h e c u t i c l e o f 1 and 4  plerocercoids. the  (3)  resembling the basal  T h i s may  process o f formation  i n d i c a t e t h a t the pore canals  are-in  i n t h e s e s m a l l worms.  O b s e r v a t i o n s on embedding and f i x a t i o n No d i f f e r e n c e s  mg  c o u l d be d e t e c t e d  techniques. between  material  embedded i n e p o n a n d t h a t embedded i n a r a l d i t e a n d b o t h were judged s a t i s f a c t o r y .  Tissue  f i x e d i n osmium  displayed  40  a t y p i c a l l y granular i s not was  possible  a property  Figure  39  nature i n e l e c t r o n micrographs but  at t h i s time to s t a t e whether t h i s o f the  f i x a t i o n or a r e s u l t of  illustrates  material.  the  i n a l l other respect  and  show t h e  38)  d i d not  result  faulty staining.  a p p e a r a n c e o f osmium f i x e d  Material f i x e d i n glutaraldehyde  i n osmium (see F i g u r e  39  granular  and  post-fixed  display this artefact  gave i d e n t i c a l r e s u l t s .  Figures  e s s e n t i a l l y i d e n t i c a l morphology of  but  38  and  mitochondria  e n d o p l a s m i c r e t i c u l u m when t i s s u e s w e r e f i x e d w i t h  different  i t  the  two  procedures. In c o n t r a s t ,  f i x a t i o n i n paraformaldehyde  and  embeddment i n M a r a g l a s r e s u l t e d i n a m o r p h o l o g y  significantly  d i f f e r e n t from t h a t d e s c r i b e d  33).  c u t i c u l a r membranes was was be  less.  The  (see  much c l e a r e r b u t  Figure the  general  the  p l a s m a membrane (see F i g u r e  36).  the  There  to the  Improved m u c o p r o t e i n  surface fixation  paraformaldehyde f i x e d t i s s u e would p o s s i b l y e x p l a i n  presence of a r e t i c u l a r c o n t e n t i n the  by  could  t h a t p a r a f o r m a l d e h y d e f i x a t i o n made  v i s i b l e a layer, p o s s i b l y mucoprotein, attached  The  of  contrast  p r o x i m a l f i b r o u s c o r e and  d i s t a l p o r t i o n were v e r y i n d i s t i n c t .  also i s reason to b e l i e v e  i n the  Detail  p l a s m a membrane s u r r o u n d i n g m i c r o t r i c h e s  v e r y c l e a r l y seen but  f i b r e s of the  above  the  electron lucid vesicles.  p r e s e n c e o f c u t i c u l a r m u c o p r o t e i n components i s i n d i c a t e d the r e s u l t s o f h i s t o c h e m i c a l  tests  (see  page  24).  41  (4)  Observations  on s t a i n i n g  Although with lead citrate, with contamination, i n technique 224).  techniques.  most o f t h e m a t e r i a l o b s e r v e d c o n s i d e r a b l e d i f f i c u l t y was a p r o b l e m more l i k e l y  was s t a i n e d experienced  the r e s u l t o f e r r o r s  t h a n o f s t a i n p r o p e r t i e s ( s e e P e a s e , 1964, page  The m o s t s a t i s f a c t o r y r e s u l t s w e r e o b t a i n e d w i t h u s e  of u r a n y l a c e t a t e and l e a d a c e t a t e s i n c e t h i s p r o d u c e d e x c e l l e n t s t a i n i n g o f membraneous p a r t i c u l a r l y v i v i d chromatin  staining  technique  components and a  (see F i g u r e 3 7 ) .  42  DISCUSSION  These r e s u l t s ,  as do t h o s e o f p r e v i o u s w o r k e r s ,  s u g g e s t t h e h y p o t h e s i s t h a t t h e c e s t o d e t e g u m e n t i s an actively metabolizing cellular and c o m p o s i t i o n .  t i s s u e of unique  structure  I t would appear t h a t the tegument i s  composed o f a n o u t e r s y n c y t i a l e n v e l o p e w h i c h e n c l o s e s t h e worm a n d i s d i r e c t l y c o n n e c t e d b y c y t o p l a s m i c b r i d g e s t o n u c l e a t e d r e g i o n s i n the parenchyma. Threadgold tegument r e s e m b l e s a  (1962) h a s r e m a r k e d  t h a t the cestode  " p e c u l i a r l y e v e r t e d g u t " and a l t h o u g h i t  w o u l d be a m i s t a k e t o d w e l l e x c e s s i v e l y on t h i s certain similarities The  analogy,  o f s t r u c t u r e and f u n c t i o n a r e a p p a r e n t .  c u t i c l e must, o f n e c e s s i t y ,  f u n c t i o n as an  absorptive  o r g a n i n t h e same manner as t h e i n t e s t i n a l e p i t h e l i u m c o n v e r s e l y i t must a l s o p r o t e c t i t s e l f d i g e s t i v e agents.  There  and  from the a c t i o n o f  i s , t h o u g h , no i n d i c a t i o n t h a t t h e  c u t i c l e i s capable of p r o d u c i n g such agents  (Read a n d  Simmons,  1963), a f a c t w h i c h must be c o n s i d e r e d i n any c o m p a r i s o n o f t h e two  tissues. The c e s t o d e m i c r o t r i c h e s h a v e b e e n c o m p a r e d t o  m i c r o v i l l i and i t has been h y p o t h e s i z e d (Threadgold, Rothman, 1963;  Race e t a l . ,  1965)  similar absorptive functions. a function,  t h a t t h e y may  1962;  perform  These w o r k e r s a l s o  suggest  i n v o l v i n g an i n t e r d i g i t a t i o n o f t h e d e n s e  distal  ends o f t h e m i c r o t r i c h e s w i t h t h e m i c r o v i l l i o f t h e h o s t ' s  c e l l s a n d e f f e c t i n g t h e worm's p o s i t i o n . has  (1962)  s u g g e s t e d t h a t t h e m i c r o t r i c h e s may h a v e a n a b r a s i v e  f u n c t i o n and thus,  destroying host c e l l s ,  nutrient concentrations The  Threadgold  provide  high  i n t h e immediate area o f the p a r a s i t e  long microthrix t y p i c a l of the s u p e r f i c i a l portions of  S. s o l i d u s w o u l d seem t o o f f e r no a d v a n t a g e f o r a n y o f t h e s e functions.  The r e l a t i v e l y  great increase i n length of the  e l e c t r o n d e n s e d i s t a l p o r t i o n w o u l d s a c r i f i c e r i g i d i t y f o r no gain i n absorptive area.  I t may b e t h a t t h e p r i m e f u n c t i o n  o f t h e m i c r o t h r i x b o r d e r o f _S. s o l i d u s i s t h e c r e a t i o n o f a microenvironment which prevents p r o t e c t i v e mucin coat. i n the observation  the d i s p e r s a l of a  Support f o r t h i s hypothesis  that a material, considered  i s found  (on t h e b a s i s  o f i t s morphology and from h i s t o c h e m i c a l evidence) t o be a m u c o p o l y s a c c h a r i d e was f r e q u e n t l y d e m o n s t r a b l e i n m i c r o g r a p h s as shown i n F i g u r e 3 3 . consider  t h i s mucin coat  I t s h o u l d b e e m p h a s i z e d t h a t I do n o t t o be i d e n t i c a l  to the thin  surface  l a y e r o f s u l p h a t e d mucoprotein which has been demonstrated histochemically.  Threadgold  (1965) d e s c r i b e d  inward  p r o j e c t i o n s o f t h e b a s e s o f some m i c r o t r i c h e s a n d r e m a r k e d t h a t they were m o r p h o l o g i c a l l y r e m i n i s c e n t o f c i l i a r y bodies.  basal  A s i n g l e micrograph o f the c u t i c u l a r surface of  _S. s o l i d u s p u b l i s h e d b y M c C a i g a n d H o p k i n s  (1965; page 268)  showed an a p p a r e n t c o n t i n u a t i o n o f t h e c e n t r a l c o r e o f microthrix fibers  into the c u t i c u l a r matrix,  p o i n t was n o t n o t e d b y t h e a u t h o r s .  although the  44  The to  c u t i c u l a r processes  S. s o l i d u s among t h e c e s t o d e s  resembling  represent  d e s c r i b e d thus f a r .  the c u t i c u l a r processes  P. p o l l a n i c o l i  (Threadgold,  a feature peculiar Structures  have been d e s c r i b e d f o r  1965) b u t t h e y a r e a p p a r e n t l y  n e i t h e r a s numerous n o r a s s t r u c t u r a l l y s i g n i f i c a n t a s reported herein. not  f u l l y developed u n t i l  infective size and  The c u t i c u l a r p r o c e s s e s  those  o f _S. s o l i d u s a r e  t h e p l e r o c e r c o i d has r e a c h e d an  ( a t l e a s t 10 mg; H o p k i n s a n d M c C a i g , 1963)  t h e y may h a v e an a d h e s i v e  function i n the f i n a l  host.  S i n c e _S. s o l i d u s h a s no f u n c t i o n a l h o l d f a s t o r g a n s t h e c u t i c u l a r processes  c o u l d f u n c t i o n i n t h i s manner b y  interdigitating with intestinal addition,  folds i n the f i n a l host.  i t w o u l d be d i f f i c u l t t o d e s c r i b e a f u n c t i o n f o r  them i n t h e s t i c k l e b a c k c o e l o m e . t h a t these area  In  Finally,  i t i s possible  s u p e r f i c i a l s t r u c t u r e s may i n c r e a s e t h e s u r f a c e  f o r a b s o r p t i o n i n a manner a n a l o g o u s t o i n t e s t i n a l The  villi.  u l t r a s t r u c t u r e o f the pore c a n a l s o f S i . s o l i d u s  described h e r e i n d i f f e r s markedly from t h a t reported by Threadgold  (1962) f o r D. c a n i n u m a n d Rothman (1963) f o r  H. d i m i n u t a .  A t v a r i a n c e w i t h my o b s e r v a t i o n s  the pore c a n a l s  o f D. c a n i n u m a p p a r e n t l y  f o r j3. s o l i d u s ,  l i e between s e c t i o n s  o f t e g u m e n t , h a v e no m i c r o t r i c h e s o r i g i n a t i n g i n t h e l u m e n and  terminate  o n t h e s u b c u t i c u l a r membrane  The  pore c a n a l s  vesicles  19 6 2 ) .  d e s c r i b e d b y Rothman (1963) a r e o b v i o u s l y  u n r e l a t e d m o r p h o l o g i c a l l y t o those represent  (Threadgold,  o f _S. s o l i d u s a n d p o s s i b l y  s t r u c t u r e s s i m i l a r t o the e l e c t r o n l u c i d ( s e e page 3 5 ) . T h a t a l l t h r e e  types  cuticular  o f "pore  canals"  45  are  r e l a t e d i s u n l i k e l y and  a c l a r i f i c a t i o n o f what a c t u a l l y  c o n s t i t u t e s a pore c a n a l must a w a i t f u r t h e r On canals  o f S.  intense  PAS  the b a s i s of m o r p h o l o g i c a l s o l i d u s may  r e a c t i v i t y and  d i s p l a y e d by  considered the  as  pore  secretory.  t o l u i d i n e blue  the  The  orthochromasia  e l e c t r o n microscope are  (non-acid) mucopolysaccharides.  the h i s t o c h e m i c a l the  e v i d e n c e the  the b a s a l o r g a n e l l e s would i n d i c a t e t h a t  droplets observed with of n e u t r a l  be  study.  pore canals  composed  In a d d i t i o n ,  a n a l y s i s demonstrated t h a t the bases  represent  surface which are  the  the o n l y areas of the c u t i c u l a r  free of o r i e n t e d negative  in close proximity  to the  of  parenchyma.  As  c h a r g e s and  such, these  are  areas  would o f f e r l e s s i n t e r f e r e n c e to the uptake o f c e r t a i n l a r g e m o l e c u l e s w h i c h m i g h t o t h e r w i s e be mucopolysaccharides. t h a t the pore c a n a l s the  pH  There i s , however, the are a c t u a l l y the  c u t i c u l a r sulphated  manner i n w h i c h t h e 1.5  and  e x c l u d e d by  c u t i c u l a r surface  stains with  B,  c o u l d be  azure A  a c i d mucopolysaccharide.  e x p l a i n e d by  although biochemical  the  a definite identification.  w o u l d be  c u t i c u l a r surface  of  The  chondroitin necessary  Biebrich scarlet staining  shown t h a t s t r o n g l y b a s i c h i s t o n e - l i k e p r o t e i n s  a l s o found a t the  at  testicular  presence of  analyses  of  The  w i t h aldehyde f u c h s i n i n d i c a t e s the presence  hyaluronidase  has  possibility  s i t e s of secretion  r e s i s t a n c e of t h i s l a y e r to d i g e s t i o n w i t h  to obtain  further  a c i d mucopolysaccharides.  a t h i n l a y e r of sulphated  sulphate  cuticular acid  and  are  through the depth  of  46  the pore c a n a l s .  Spicer  (1962) h a s shown t h a t t h i s  type o f  protein i s often associated with a c i d mucopolysaccharides. Spicer  (1962) h a s a l s o i n d i c a t e d t h a t t h e weak a z u r o p h i l i a o f  c e r t a i n i n t e s t i n a l g o b l e t - c e l l sulphomucins masking moiety. might  o f sulphate  may b e due t o  esters by combination with  a basic  A sulphomucin-basic p r o t e i n complex o f t h i s  type  e x p l a i n the l a c k o f a z u r o p h i l i a d i s p l a y e d by the b a s a l  o r g a n e l l e s o f the pore c a n a l s . hypothesis  i s provided  Further  strength f o r this  by the observation  t h a t pore  canal  r e a c t i v i t y w i t h B i e b r i c h s c a r l e t may h a v e i n c l u d e d t h e basal organelle.  A complex o f t h i s type would  explain the  v a r i a b l e metachromasy d i s p l a y e d b y t h e most s u p e r f i c i a l o f t h e c u t i c u l a r m a t r i x o f many p l e r o c e r c o i d s . f u r t h e r work w i l l be n e c e s s a r y t o c l a r i f y  this  1-2 p .  However, possibility.  If the s u p e r f i c i a l portion of the c u t i c u l a r  matrix  does c o n t a i n a s u l p h o m u c i n - b a s i c p r o t e i n c o m p l e x i t i s apparent t h a t free sulphate extreme s u r f a c e  of this  groups  layer.  a r e found o n l y on t h e  Monne  (1959) h a s d e m o n s t r a t e d  the presence o f a c i d mucopolysaccharides surfaces  on t h e c u t i c u l a r  o f s e v e r a l tapeworms a n d h a s s u g g e s t e d a p o s s i b l e  r o l e a s h o s t enzyme i n h i b i t o r s (1963) h a s d e t e c t e d  f o rthese substances.  a similar layer of acid  Crompton  mucopolysaccharides  on t h e s u r f a c e o f t h e a c a n t h o c e p h a l a n worm P o l y m o r p h u s minutus.  Bogitsh  (1963) a l s o a s s i g n s  a protective function  to the l a y e r o f a c i d mucopolysaccharides c u t i c l e o f Hvmenolepis  microstoma.  o b s e r v e d on t h e  However, W a i t z a n d  47  Schardein  (1964) do  not  i n t h e i r s t u d y o f H. and of  Dipylidium S.  solidus  stickleback  n a n a , H.  caninum.  t h i s a c i d mucopolysaccharide diminuta, Hydatigera  I f the  rather  function  than i n the  colelomic  c a v i t y of  intestinal habitat  coat  i t is  t h i s coat i s already present i n  w h i c h e x i s t s i n the  coat  taeniaeformis  a c i d mucopolysaccharide  does i n f a c t have a p r o t e c t i v e  o f some i n t e r e s t t h a t plerocercoid  report  the the  of  the  mature  form. I t may  w e l l be  that  the  inhibitory activity  the m u c o p r o t e i n l a y e r v a r i e s w i t h  the  pH  medium, i . e . , f r o m a p p r o x i m a t e l y n e u t r a l  of  of  the  surrounding  i n the  stickleback  coelomic c a v i t y to a c i d i n the b i r d i n t e s t i n e . I f such a v a r i a t i o n i n the of  the  sulphomucin-basic protein  also explain  the  are  ( a c i d and  functional  stickleback  not  I t i s probable that  growth of  the  c o e l o m e , a t a pH w h e r e t h e  by mucoproteins c o u l d  be  with  cuticular  plerocercoid  others) in  the  of  life  Since of  nutrients  t h e worm  the  t h e s e enzyme s y s t e m s  tolerated.  I t w o u l d , p e r h a p s , be by  p r o f i t a b l e t o m e n t i o n some  other properties  displayed  possessed by  cestode c u t i c l e .  the  could  sulphomucin-protein  short adult  absorptive functions  the  possibly  exhibit inhibitory action.  absorbed d u r i n g the  n e g a t i o n o f any  such a complex  a l k a l i n e p h o s p h a t a s e s , and  d u r i n g the  complex might not are  complex d i d e x i s t , t h i s  concurrent existence of  s u r f a c e c u t i c u l a r enzymes. enzymes  enzyme i n h i b i t o r y a c t i v i t y  polyelectrolytes At  a c i d pH's  of  the  (< pH  type 6)  48  p o l y a c i d s s u c h as p o l y a c r i l a t e e x i s t i n a r e l a t i v e l y f o r m due t o t h e numerous h y d r o g e n b o n d s p r e s e n t c a r b o x y l i c groups.  contracted  between t h e  I f some o f t h e a c i d i c g r o u p s a r e i o n i z e d  by t h e a d d i t i o n o f a base, r e p u l s i v e f o r c e s develop between the charged c a r b o x y l groups and t h e c h a i n tends t o u n f o l d and lengthen  (Katchalsky,  1964).  Such a phenomenon w o u l d  probably  enhance t h e i n h i b i t o r y a c t i v i t y o f a p o l y a c i d b y i n c r e a s i n g t h e number o f e x p o s e d c h a r g e d g r o u p s , a n i n c r e a s e w h i c h w o u l d b e dependent on t h e s u r r o u n d i n g a l s o may h a v e s e p a r a t e 19 6 4 ) .  pH.  B a s i c and a c i d p o l y e l e c t r o l y t e s  enzyme i n h i b i t i n g a c t i v i t i e s  F o r example, t h e a c t i o n o f p e p s i n  (Katchalsky,  i s i n h i b i t e d by  b a s i c polymers and r e s t o r e d b y t h e sulphomucin h e p a r i n . the o t h e r hand, the a c t i v i t i e s o f h y a l u r o n i d a s e  On  and r i b o n u c l e a s e  are i n h i b i t e d by p o l y a c i d s and t h e i r a c t i v i t y i s r e s t o r e d by polybases  (Katchalsky,  1964).  The h i s t o c h e m i c a l p r o p e r t i e s o f t h e b u l k o f t h e cuticular matrix  indicate that this region also  contains  s i g n i f i c a n t q u a n t i t i e s of a c i d mucopolysaccharides hyaluronic acid.  I t i s l i k e l y that hyaluronate,  probably  a s s o c i a t e d w i t h p r o t e i n , forms t h e c u t i c u l a r ground w i t h i n which the c u t i c u l a r v e s i c l e s are l o c a t e d . hyaluronate  substance  A  m a t r i x c o u l d f u n c t i o n i n b o t h s t r u c t u r a l and  physiological capacities.  The l a r g e h y d r o d y n a m i c  volume and accompanying t u r g e s c e n c e hyaluronate  probably  specific  characteristic of  w o u l d endow t h e c u t i c l e w i t h a c e r t a i n r i g i d i t y  49  as w e l l as p r o v i d i n g a c o n t r o l over random d i f f u s i o n o f l a r g e molecules.  I t has a l s o been h y p o t h e s i z e d (see Rogers, 1961)  t h a t h y a l u r o n a t e ( i n a d d i t i o n to o t h e r mucopolysaccharides) may f u n c t i o n as an exchange  r e s i n and r e g u l a t e the flow and  concentration of cations. This i s to my knowledge the f i r s t demonstration o f a c i d mucopolysaccharides i n the c u t i c u l a r m a t r i x . c o u l d d e t e c t no t o l u i d i n e b l u e metachromasia  Waitz  (1963)  i n the c u t i c l e o f  a d u l t s and l a r v a e o f Hydaticrera t a e n i a e f o r m i s ; b u t he d i d s p e c u l a t e t h a t the observed o r t h o c h r o m a t i c s t a i n i n g below pH 4.0 might i n d i c a t e the presence o f an a c i d mucopolysaccharide. Various phosphatases have been r e p o r t e d from many types o f helminths (Ma, 19 64).  In the m a j o r i t y o f cases both  a c i d and a l k a l i n e phosphatases have been d e t e c t e d though, i n some instances  (Yamao, 1952; Lee and T a c h e l l ,  a c i d phosphatase l e v e l s c o u l d be shown.  1964), no s i g n i f i c a n t Alkaline  phosphatase  appears to be an almost u n i v e r s a l c o n s t i t u e n t o f the helminth coat.  Studies by Erasmus  (1957a, 1957b) on the phosphatases o f  Taenia p i s i f o r m i s , Moniezia expansa and C y s t i c e r c u s  tenuicollis  agree c l o s e l y w i t h the r e s u l t s p r e s e n t e d f o r _S. s o l i d u s . should be borne i n mind t h a t a spectrum o f phosphatase  It  isozymes  may e x i s t and a l l may be demonstrated by one h i s t o c h e m i c a l reaction.  Ma  (1964) has d e s c r i b e d t h r e e a c i d  isozymes from the t i s s u e o f C l o n o r c h i s  phosphatase  sinensis.  A g r e a t d e a l o f s p e c u l a t i o n has c e n t e r e d on the r o l e o f h e l m i n t h c u t i c u l a r phosphatases.  Bullock  (1949, 1958)  50  has c l a i m e d t h a t phosphatases  i n the acanthocephalan  cuticle  are concerned w i t h the a b s o r p t i o n o f f o o d s t u f f s o r w i t h e l i m i n a t i o n of waste  products.  Erasmus  t h a t a c i d a n d a l k a l i n e p h o s p h a t a s e s may r e l e a s e o f phosphate  (1957a, 1957b) s u g g e s t s be i n v o l v e d i n t h e  f o r carbohydrate metabolism or  f o r p h o s p h o r y l a t e d passage o f m a t e r i a l s through the Lewert and D u s a n i c  the  perhaps cuticle.  (1960) h a v e shown t h a t t h e i n h i b i t i o n  a l k a l i n e phosphatase  a c t i v i t y i n Schistosoma mansoni by  of a  diamino-dibenzyl-alkane i s c o r r e l a t e d w i t h a decrease i n i n v i t r o glucose uptake. Ponz a n d L a r r a l d e  S i m i l a r w o r k h a s b e e n done b y  (1952) u s i n g p h l o r i z i n as an enzyme  However, J e r v i s e t _ a l . ,  inhibitor.  (1956) h a v e d e m o n s t r a t e d t h a t g l u c o s e  u p t a k e i s i n h i b i t e d b y much l o w e r c o n c e n t r a t i o n s o f  phlorizin  than are necessary f o r the i n a c t i v a t i o n of a l k a l i n e  phosphatase.  S i m i l a r r e s u l t s p u b l i s h e d by P h i f e r  that  phosphates  (19 60)  indicate  are not necessary f o r glucose metabolism.  The  r e s u l t s o f t h i s s t u d y do n o t j u s t i f y a n y f u r t h e r comment o n the f u n c t i o n s o f c u t i c u l a r The  phosphatases.  d i s t r i b u t i o n of cuticular  lipids  i n _S. s o l i d u s  agrees s u b s t a n t i a l l y w i t h t h a t d e s c r i b e d by Waitz H. t a e n i a e f o r m i s .  (1963) f o r  S i n c e t h e Sudan b l a c k s t a i n i n g o f t h e  _S. s o l i d u s c u t i c l e p r e s u m a b l y i n d i c a t e s t h e p r e s e n c e o f sphingolipids,  the observed decrease i n s t a i n i n t e n s i t y i n  l a r g e p l e r o c e r c o i d s may  r e f l e c t a decrease w i t h growth i n  t h e q u a n t i t y o f c u t i c u l a r membranes. that a substantial  l i p i d c o m p o n e n t may  Another possibility i s be c o m p l e x e d  with  51  c u t i c u l a r p r o t e i n s i n the r o d - l i k e c u t i c u l a r v e s i c l e s s i n c e the decrease i n l i p i d s t a i n i n g w i t h growth i s concomitant w i t h t h a t o f the p r o t e i n s t a i n and the q u a n t i t y o f r o d - l i k e v e s i c l e s decreases w i t h an i n c r e a s e i n p l e r o c e r c o i d s i z e .  A further  i n d i c a t i o n t h a t the r o d - l i k e v e s i c l e s may c o n t a i n a substance i s found i n the o b s e r v a t i o n  t h a t my h i g h r e s o l u t i o n  micrographs r e v e a l the presence o f a l a t t i c e - l i k e resembling  proteinaceous  matrix  t h a t seen i n c e r t a i n c r y s t a l l i n e c e l l u l a r i n c l u s i o n s  (see Fawcett, 1966). I f , as suggested above  (see Page 40),  the e l e c t r o n  l u c i d c u t i c u l a r v e s i c l e s c o n t a i n mucopolysaccharides the i n c r e a s e i n number o f these v e s i c l e s i n l a r g e r worms would e x p l a i n the i n c r e a s e i n c u t i c u l a r PAS s t a i n i n g .  I t should  be emphasized t h a t s i n c e h y a l u r o n i c a c i d does not give the PAS r e a c t i o n (Barka and Anderson, 19 63)  the change i n c u t i c u l a r  r e a c t i v i t y must r e f l e c t a change i n some other c u t i c u l a r carbohydrates.  An i n c r e a s e i n the c u t i c u l a r PAS response o f  a d u l t H. t a e n i a e f o r m i s by Waitz  over t h a t o f l a r v a l forms was r e p o r t e d  (1963) although t h i s i n c r e a s e was c o n f i n e d to the  surface region.  However, the i n c r e a s e i n c u t i c u l a r  carbohydrate composition  r e p o r t e d h e r e i n must be regarded w i t h  some c a u t i o n s i n c e the i n t e n s e PAS r e a c t i v i t y d i s p l a y e d by the c u t i c l e o f l a r g e r worms d i d n o t c o n s i s t e n t l y occur when f u r t h e r specimens were examined. This r e p o r t i s a p p a r e n t l y  the second to i n d i c a t e the  presence o f RNA i n the cestode c u t i c l e  (Rothman,  personal  52  communication). c u t i c u l a r RNA s t a i n i n g and H.  However, B o g i t s h  i n H. Waitz  c e r t a i n questions activities.  microstoma a f t e r methyl (1963) d i d n o t  taeniaeformis.  The on  Lumsden  (1965) h a s  occur i n the c u t i c l e .  organelles and  poses  shown  autoradiographically  incorporated  i n the  to the c u t i c l e .  tegumental  I t w o u l d be  i f any,  On m o r p h o l o g i c a l  i s no  grounds  and  i n d i c a t i o n of ribosome-bearing c u t i c u l a r  the c u t i c u l a r m i t o c h o n d r i a  noted t h a t the  are  comparatively  glycogen deposits, which  It  l o c a t e d a t some d i s t a n c e On  the b a s i s o f the  l i k e l y t h a t the c u t i c l e  I t may  i t s cellular  be,  from the  i n v o l v e d i n the  cuticle. i t seems  a c t i v e s y n t h e t i c a l l y but  components f r o m the  production  are  available information  i s not  however, t h a t the  should  presumably  s e r v e as an e n e r g y s o u r c e f o r s y n t h e t i c a c t i v i t i e s  receives  of  synthesis  located basally i n large plerocercoids.  a l s o be  in  c u t i c u l a r s y n t h e t i c a c t i v i t i e s w o u l d seem u n l i k e l y  since there  few  RNA  the nature of c u t i c u l a r s y n t h e t i c  then transported  extensive  any  green-pyronin  attempt to detect  i n t e r e s t to determine to what e x t e n t , can  find  p r e s e n c e o f c u t i c u l a r RNA  t h a t l a b e l l e d amino a c i d s a r e c e l l s and  (1963) d i d n o t  tegumental  c u t i c u l a r mitochondria  o f ATP  t r a n s p o r t o f n u t r i e n t s i n t o the  cells. are  n e c e s s a r y f o r the  tegument from the  rather  active  external  medium. Histochemically small plerocercoids At  and  morphologically  a p p e a r s t o be  the u l t r a s t r u c t u r a l l e v e l the  the c u t i c l e  i n a state of c u t i c l e o f 1-4  of  transition. mg  plerocercoids  53  i s c h a r a c t e r i z e d b y l a r g e numbers o f m i t o c h o n d r i a , w h i c h appear t o be i n a s t a t e o f d e g e n e r a t i o n .  many o f  The  o f t h e c u t i c u l a r v e s i c l e s appear t o be p r i m a r i l y  contents  proteinaceous,  i n agreement w i t h t h e h i s t o c h e m i c a l c h a r a c t e r i s t i c s o f t h e worm.  In these  s m a l l worms no c u t i c u l a r p r o c e s s e s  c a n be  seen and there a r e i n d i c a t i o n s t h a t pore canals a r e i n t h e process  of formation  carbohydrate  ( s e e Page 39).  The c u t i c u l a r  c o n s t i t u e n t s appear t o c o n s i s t o f a b a s a l  concentration of hyaluronic acid,a layer of s i a l i c c o n t a i n i n g mucin and a s u r f a c e l a y e r o f With p r o g r e s s i n g development  acid-  sulfomucins. ( y 10 mg) t h e c u t i c l e  becomes e l a b o r a t e l y s t r u c t u r e d a s t h e c u t i c u l a r  processes  and  a r e now  pore canals a r e developed.  The m i t o c h o n d r i a  b a s a l l y l o c a t e d a n d l a r g e numbers o f e l e c t r o n l u c i d v e s i c l e s are present.  A s shown b y h i s t o c h e m i c a l e x a m i n a t i o n  this  d i f f e r e n t i a t i o n i s accompanied by a compaction o f s u r f a c e sialomucins matrix.  a n d a n i n c r e a s e i n t h e PAS r e a c t i v i t y o f t h e  The i n c r e a s e i n PAS r e a c t i v i t y i s a c c o m p a n i e d  by a d i m u n i t i o n i n the l i p o i d - p r o t e i n content be  a n d may  r e l a t e d t o t h e change i n t h e r e l a t i v e c o n c e n t r a t i o n s o f  cuticular  vesicles. I t m u s t b e a s s u m e d t h a t many of t h e a b o v e c h a n g e s  are,  i n some manner, i n v o l v e d i n t h e p r e p a r a t i o n o f t h e  plerocercoid f o rthe short adult existence The  problem o f i n t e r p r e t i n g these  i n the b i r d gut.  changes i s c o m p l i c a t e d ,  however, b y t h e f a c t t h a t t h e u l t i m a t e c u t i c u l a r s t r u c t u r e i s  54  only a t t a i n e d long a f t e r the p l e r o c e r c o i d i s i n f e c t i v e . I t may b e t h a t t h e d i f f e r e n t i a t i o n o f t h e p l e r o c e r c o i d i s d i v i d e d i n t o two p h a s e s ;  (1) a n i n i t i a l  period of transition  from t h e p r o c e r c o i d , d u r i n g w h i c h t h e d e f e n s i v e mechanisms r e q u i r e d f o r s u r v i v a l i n the f i n a l h o s t a r e e l a b o r a t e d , and (2) a much l o n g e r p h a s e i n w h i c h t h e p l e r o c e r c o i d i s m o d i f i e d to permit prolonged coelom.  I t would appear from the present  morphological phase.  growth and s u r v i v a l i n the s t i c k l e b a c k study t h a t the major  changes o c c u r d u r i n g t h e second  post-infective  C l e a r l y , a more d e t a i l e d s t u d y o f t h e m o r p h o l o g i c a l  and h i s t o c h e m i c a l e v e n t s  accompanying t h e f i r s t phase o f  d i f f e r e n t i a t i o n i s r e q u i r e d b e f o r e any judgement o f t h i s h y p o t h e s i s c a n b e made.  55  SUMMARY  1.  T h i s work demonstrates t h a t the p l e r o c e r c o i d of but  Schistocephalus  2.  s o l i d u s c o n s i s t s o f an o u t e r  c e l l u l a r region  cytoplasmic  sub-cuticular  The  pore c a n a l s  The  tegumental c e l l s  previously described  pore c a n a l s  3.  P a p i l l a - l i k e c u t i c u l a r p r o c e s s e s on have been d e s c r i b e d  suggested f o r these s t r u c t u r e s .  the  c u t i c u l a r processes are  not  external that  capacity.  the c u t i c u l a r  surface  absorptive  I t has  functions  b e e n shown  p r e s e n t i n the  that  smallest  same t i m e  infective.  c u t i c u l a r matrix  that increased  other  the  r a t h e r appear a t about the  Changes i n t h e h i s t o c h e m i c a l of the  differ  grounds  b o t h a d h e s i v e and  are  as t h e worm becomes 4.  and  for  with  function i n a secretory  forms s t u d i e d b u t  in  o f S_. s o l i d u s a l l o w c e r t a i n  I t i s s u g g e s t e d on m o r p h o l o g i c a l  t h e s e c e l l s may  lying  o f _S. s o l i d u s h a v e b e e n shown t o  parenchymal c e l l s a d i r e c t connection medium.  by  parenchyma.  i n s t r u c t u r e f r o m any cestodes.  anucleate  (the c u t i c l e ) w h i c h i s c o n n e c t e d  tubes to nucleated  the  tegument  and  ultrastructural  have been d e s c r i b e d .  composition  I t i s probable  p l e r o c e r c o i d growth i s accompanied by  a  c h a n g e i n c u t i c u l a r c o m p o s i t i o n f r o m a more p r o t e i n a c e o u s s t a t e t o one 5.  which i s l a r g e l y  carbohydrate.  A surface  s u l p h o m u c i n - b a s i c p r o t e i n complex has  described  and  partially characterized.  Possible  been functions  56  have been suggested f o r t h i s 6.  RNA  layer.  as w e l l as a c i d a n d a l k a l i n e p h o s p h a t a s e s w e r e  found i n the c u t i c l e and t h e p o s s i b l e  significance of  these substances i n c u t i c u l a r synthetic  activities i s  considered. 7.  I t i s suggested that p l e r o c e r c o i d c u t i c u l a r d i f f e r e n t i a t i o n may (1)  (2)  c o n s i s t o f two  phases:  an i n i t i a l  phase  elaborated  to allow  intestine,  and  a s e c o n d phase  i n which various  s u r v i v a l i n the  vertebrate  i n which the p l e r o c e r c o i d i s  to p e r m i t p r o l o n g e d growth i n the coelom.  mechanisms a r e  modified  stickleback  L I S T OF ABBREVIATIONS  Abbreviations  f o r structures:  BF - b a s a l  fibrils  of microthrix  CP - c u t i c u l a r p r o c e s s DP - d i s t a l p o r t i o n o f m i c r o t h r i x ED - e l e c t r o n - l u c i d d r o p l e t s o f p o r e ER - e n d o p l a s m i c  canal  reticulum  F - fibrous apical portion of microthrix M - mitochondrion MB - m y e l i n f i g u r e MU - m u c o u s - l i k e  material  MV - c u t i c u l a r v e s i c l e s p r e s u m e d t o b e o f mitochondrial o r i g i n N - nucleus NL -  nucleolus  PC - p o r e  canal  PM - p l a s m a membrane SM - s u b c u t i c u l a r membrane V - velum r e g i o n o f c u t i c l e VR - v a c u o l a t e d r e g i o n o f c y t o p l a s m i c bridge between e x t e r n a l and i n t e r n a l l e v e l s  Abbreviations  f o r f i x a t i v e s used i n l i g h t  studies: FS  -  FC  -  formol-saline formol-calcium  microscope  58  FA -  formol-alcohol  CPC/F - . 5 % CPC i n 1 0 % f o r m a l i n Acet. - cold  acetone  A l l w e i g h t s a r e e x p r e s s e d i n mg,  e g . 'FS 138' i n d i c a t e s t h a t  t h e m i c r o g r a p h i s o f a 139 mg worm w h i c h h a s b e e n f i x e d i n formol-saline.  The a b b r e v i a t i o n s a n d methods  f o r s t a i n s are given i n s e c t i o n E of materials  (pages 4 - 7 ) .  59  PLATE I F i g u r e 1.  Carnoy-fixed. 5 mg TB. 850X. The o r t h o c h r o m a t i c a l l y s t a i n i n g s u r f a c e l a y e r and the n o n - s t a i n i n g m i c r o t h r i x border are v i s i b l e . The p o r t i o n shown i s t y p i c a l o f the c u t i c l e o f v e r y s m a l l worms.  F i g u r e 2.  FS 138. AA 4.0. 850X. The e n t i r e c u t i c l e i s metachromatic w i t h the most i n t e n s e r e a c t i o n a t the c u t i c u l a r s u r f a c e . The b a s a l o r g a n e l l e o f the pore canal i s c l e a r l y orthochromatic. Note a l s o the c u t i c u l a r processes and the d i f f e r e n c e i n the t h i c k n e s s o f the s u p e r f i c i a l and v e l a r p o r t i o n s o f the c u t i c l e .  F i g u r e 3.  FC 101. AB-PAS. 225X. Note the r e l a t i v e l y i n t e n s e a l c i a n b l u e s t a i n i n g i n the a n t e r i o r p o r t i o n o f the superficial cuticle. The s u b c u t i c u l a r membrane i s PAS p o s i t i v e .  F i g u r e 4.  FA 26. AB-PAS. 1,365X. The r e g i o n shown i n c l u d e s p o r t i o n s o f two segments. In worms o f t h i s weight there i s l i t t l e d i f f e r e n c e i n the c u t i c u l a r t h i c k n e s s o f the velum and the s u p e r f i c i a l p o r t i o n . Three c u t i c u l a r l a y e r s a r e i n d i c a t e d . I and I I I are PAS p o s i t i v e w h i l e I I i s AB p o s i t i v e .  F i g u r e 5.  FS 90. AF-AB. 135X. The s u p e r f i c i a l AF p o s i t i v e l a y e r and the AB p o s i t i v e l a y e r can be seen.  F i g u r e 6.  FS 212. AF-AB. 850X. The AB p o s i t i v e l a y e r i s d i f f i c u l t to d i s t i n g u i s h as a separate e n t i t y i n worms o f t h i s s i z e .  F i g u r e 7.  FS 65. TB. 1,080X. S e v e r a l pore c a n a l s can be seen r e c e i v i n g processes from parenchymal c e l l s . Note a l s o the orthochromatic s u r f a c e l a y e r and t h e d i f f e r e n c e i n o r i e n t a t i o n o f the tegumental c e l l s beneath the velum and beneath the s u p e r f i c i a l c u t i c l e .  F i g u r e 8.  FS 138. MBPB. 850X. Note the r e l a t i v e l y i n t e n s e s t a i n i n g o f the s u r f a c e and o f the matrix immediately beneath the sub-surface l a y e r . The bulk o f the c u t i c u l a r matrix has s t a i n e d l i g h t l y w i t h t h i s method.  60  PLATE I I Figures  Figure  9, 10 a n d 1 1 . CPC/F 1 7 1 . AA 1.5, 2.5 a n d 4.0. 1 0 8 X . A t pH's o f 1.5 a n d 2.5 ( F i g u r e s 9 a n d 10) o n l y t h e s u r f a c e o f t h e c u t i c l e r e a c t s w i t h a z u r e A. When t h e pH i s r a i s e d t o 4.0 ( F i g u r e 11) t h e e n t i r e c u t i c u l a r matrix reacts with the s t a i n . 12. F S 1 1 0 . AA 4.5. 340X. A t t h i s pH t h e e n t i r e c u t i c l e i s s t r o n g l y metachromatic. The i n d i c a t e d e x t r a c u t i c u l a r mucin reacted c o n s i s t e n t l y w i t h a l c i a n b l u e when t h a t m e t h o d was a p p l i e d .  Figures  13 a n d 1 4 . F S 1 2 3 . TB a n d TB a f t e r h y a l u r o n i d a s e digestion. 108X. T r e a t m e n t w i t h h y a l u r o n i d a s e removed most b u t n o t a l l o f t h e c u t i c u l a r metachromas i a .  Figures  15 a n d 1 6 . CPC/F 1 7 1 . TB a n d TB a f t e r R N A s e d i g e s t i o n , 85X. When RNA'se d i g e s t i o n was e m p l o y e d t h e b u l k o f t h e c u t i c u l a r m e t a c h r o m a s i a was r e m o v e d (compare w i t h F i g u r e 1 4 ) . The r e a c t i v i t y o f t h e s u r f a c e was .not, however, i m p a i r e d . 1  61  PLATE I I I Figures Figure  17 a n d 1 8 . F S 1 1 0 . MGP-and M G P - a f t e r w i t h RNA'se. 108X. 19.  F i g u r e 20. Figures  digestion  A c e t o n e - f i x e d m a t u r e worm. A z o - c o u p l i n g for a l k a l i n e phosphatase. 108X. A c e t o n e - f i x e d m a t u r e worm. a c i d phosphatase. 108X.  method  Gomori method f o r  2 1 , 22 a n d 2 3 . FA 30, F S 3 5 , a n d F S 9 0 . SBB m e t h o d f o r bound l i p i d s . 85X. N o t e t h e d e c r e a s e d l i p i d staining with increased size. A l s o note the intense staining of the calcareous corpuscles i n F S - f i x e d worms.  F i g u r e 24.  FC 1 0 1 . SBB. 85X. A c o m p a r i s o n o f t h i s m i c r o g r a p h w i t h t h a t i n F i g u r e 23 w i l l emphasize the improvement i n l i p i d s t a i n i n g w h i c h r e s u l t e d from f o r m o l - c a l c i u m f i x a t i o n .  62  PLATE IV F i g u r e 25.  S e c t i o n o f c u t i c u l a r s u r f a c e s o f velum and superf i c i a l r e g i o n o f a 22 mg p l e r o c e r c o i d . Note absence o f c u t i c u l a r processes a t velum s u r f a c e and presence o f mucous-like m a t e r i a l . Glutaraldehyde f i x e d , 0 04 p o s t - f i x e d ( G l u t . - 0 0 4 ) . Embedded i n A r a l d i t e ( A r ) . 22,000X. S  Figure  2 6.  S  C u t i c u l a r s u r f a c e o f a 10 mg p l e r o c e r c o i d showing s u r f a c e plasma membrane and f i b r i l l a r core o f basal portion of microtriches. 0 04 f i x e d . Embedded i n Epon (Ep). 53,000X. S  F i g u r e 27,  S e c t i o n through d i s t a l p o r t i o n o f m i c r o t r i c h e s o f a 100 mg p l e r o c e r c o i d i l l u s t r a t i n g electon-dense f i b r o u s i n t e r i o r , o u t e r e l e c t r o n - l u c i d r e g i o n and e x t e r n a l plasma membrane. Paraformaldehyde f i x e d , 0 04 p o s t - f i x e d . (Par-0 04) Embedded i n Maraglas (Mar). 127,000X. S  F i g u r e 28,  S  Oblique s e c t i o n o f the s u r f a c e o f a 136 mg p l e r o c e r c o i d i l l u s t r a t i n g c u t i c u l a r v e s i c l e s and m i c r o t r i c h e s . Note c i r c u l a r shape o f b a s a l p o r t i o n o f m i c r o t r i c h e s . G l u t . - 0 0 4 . A r . 24,000X. S  F i g u r e 29,  S e c t i o n through the c u t i c l e o f a 94 mg p l e r o c e r c o i d showing p o r t i o n s o f a pore c a n a l , m y e l i n f i g u r e s and c u t i c u l a r m i t o c h o n d r i a . Note a g g r e g a t i o n o f e l e c t r o n - l u c i d d r o p l e t s a t base o f pore c a n a l and the m i c r o t r i c h e s o r i g i n a t i n g from the w a l l s . Glut.-O 04. A r . 6,000X. B. Photographic enlargement o f a m y e l i n f i g u r e from the c u t i c l e o f a 10 mg p l e r o c e r c o i d . 57,000X. s  63  PLATE V Figure  30.  B a s a l r e g i o n o f a p o r e c a n a l f r o m an 18 mg p l e r o c e r c o i d showing e l e c t r o n - l u c i d d r o p l e t s and m u c o u s - l i k e m a t e r i a l i n lumen. Glut.-0 0 . Ar. 12,000X. s  Figure  31.  D e t a i l of the e l e c t r o n - l u c i d d r o p l e t s observed i n t h e p o r e c a n a l o f a 27 mg p l e r o c e r c o i d . 0 0 . Ep. 35,000X.. S  Figure  32.  4  4  S e c t i o n t h r o u g h t h e s u b c u t i c u l a r membrane o f a 10 mg p l e r o c e r c o i d i l l u s t r a t i n g t h e v a c u o l a t e d r e g i o n s of the c y t o p l a s m i c b r i d g e s . Note t h a t the mitochondria of the cytoplasmic b r i d g e s are l a r g e r than those of the c u t i c l e . 0 0 4 . Ep. 13,000X. S  64  PLATE V I Figure  33.  S e c t i o n t h r o u g h v e l u m and s u p e r f i c i a l c u t i c l e o f a 100 mg p l e r o c e r c o i d . Note the c u t i c u l a r v e s i c l e s and the p r e s e n c e o f e x t r a c u t i c u l a r m u c o u s - l i k e m a t e r i a l among t h e v e l u m m i c r o t r i c h e s . Par.-0 04. Mar. 12,000X. S  Figure  34.  B a s a l r e g i o n o f t h e c u t i c l e o f a 1 mg p l e r o c e r c o i d s h o w i n g d e t a i l o f v e s i c l e s b e l i e v e d t o be o f mitochondrial origin. Glut.-0 04. Ep. 23,000X. S  65  PLATE VII F i g u r e 35.  S e c t i o n through the c u t i c l e o f a 1 mg p l e r o c e r c o i d i n which l a r g e v e s i c l e s can be seen i n the c u t i c l e . Note the absence o f c u t i c u l a r processes. Glut.-0 04. Ep. 9,000X. S  F i g u r e 36.  D e t a i l of a m i c r o t h r i x . Note the f i b r o u s nature o f the e l e c t r o n - d e n s e d i s t a l p o r t i o n and the presence o f a plasma membrane. M a t e r i a l t h a t i s assumed to be p a r t o f a mucoprotein s u r f a c e c o a t can a l s o be seen. Par.-0 04. Mar. 135,000X. S  F i g u r e 37.  D e t a i l o f t y p i c a l parenchymal c e l l s . Note the e x t e n s i v e endoplasmic r e t i c u l u m and the t y p i c a l n u c l e a r s t r u c t u r e . 4 mg p l e r o c e r c o i d . Glut.-0 C>4. Ep. 18,000X. S  66  PLATE V I I I Figure  38.  An u n u s u a l e n d o p l a s m i c r e t i c u l u m f o r m a t i o n i n c e l l s o f the. p a r e n c h y m a o f a 136 mg p l e r o c e r c o i d . Glut.-0 94. A r . 35,000X. S  Figure  39.  C e l l u l a r o r g a n e l l e s f r o m a 161 mg p l e r o c e r c o i d . 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On the e x t e r n a l c u t i c l e s of v a r i o u s  helminths  and t h e i r r o l e i n the h o s t - p a r a s i t e r e l a t i o n s h i p . Ark.  Zool.,  7.:  343-3  58.  and L . T. THREADGOLD.  PANTELOURIS, E. M.  1963.  system o f the a d u l t F a s c i o l a h e p a t i c a .  The La  excretory Cellule,  64:63-67. PEARSE, A. G. E.  1961.  Applied. PEASE, D a n i e l C.  J . A. C h u r c h i l l , 1964.  Microscopy. PHIFER, K.  Histochemistry,  1960.  London.  H i s t o l o g i c a l Techniques f o r E l e c t r o n Academic Press, New  York and  London.  Permeation and membrane t r a n s p o r t i n animal  parasites: diminuta.  T h e o r e t i c a l and  the a b s o r p t i o n o f glucose by Hymenolepis J . P a r a s i t . , 46:51-62.  PONZ, F. and J . LARRALDE. t i o n o f sugars  1952.  R e l a t i o n of s e l e c t i v e  and some enzyme i n h i b i t o r s .  Espan. F i s i o l . , 8_:71.  absorp-  Rev.  71  QUINTARELLI, G.  a n d M.' C.  DELLOVO.  1965.  The  chemical  histochemical properties of a l c i a n blue.  and  IV.  Further  s t u d i e s on t h e m e t h o d s f o r t h e i d e n t i f i c a t i o n o f glycosaminoglycans.  Histochemie,  RACE, G e o r g e J . , J o h n E. LARSH, G e r a l d W. MARTIN.  1965.  acid  5_: 1 9 6 - 2 0 9 . ESCH, a n d  James  A study of the l a r v a l stage  M u l t i c e p s s e r i a l i s by e l e c t r o n microscopy.  H.  of J.  Parasit.,  51:364-369. READ, C.  P. and  J . E.  SIMMONS.  o f tapeworms. ROGERS, H.  J. In:  1961. The  1963.  P h y s i o l . Rev.,  The  s t r u c t u r e and  tissue  (Ed. F.  function of  C l a r k and  Cambridge U n i v e r s i t y P r e s s , ROTHMAN, A l v i n H.  1963.  tapeworms: diminuta.  Grant).  London.  Am.  microsc.  D o n a l d L. L E E .  studies of  Soc,  1963.  82:22-30. Histochemical  of dehydrogenase a c t i v i t y i n the  of cestodes.  E x p t l . P a r a s i t o l o g y , 14(3):333-336.  1946.  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SPURLOCK, B. O., V. C. KATTINE a n d J . A . FREEMAN. 1 9 6 3 . T e c h n i c a l m o d i f i c a t i o n s i n Maraglas embedding. J. Cell Biol., SZIRMAI, J o h n A.  1963.  17:203-207. Q u a n t i t a t i v e approaches i n the  histochemistry of mucopolysaccharides.  J . Histochem.  Cytochem., l l ; 2 4 - 3 3 . THREADGOLD, L . T.  1962. An e l e c t r o n m i c r o s c o p e study  of the  tegument and a s s o c i a t e d s t r u c t u r e s o f D i p y l i d i u m caninum.  Quart. J . M i c r .  THREADGOLD, L. T.  S c i . , 103:135-140.  1965. An e l e c t r o n m i c r o s c o p e s t u d y  of the  tegument and a s s o c i a t e d s t r u c t u r e s o f P r o t e o c e p h a l u s pollanicoli. WAITZ, J . A l l a n .  Parasitology,  1963. H i s t o c h e m i c a l  Hydatigera  taeniaeformis,  55;467-472. studies o f the cestode  Batsch,  1786.  J. Parasit.,  49:73-80. WAITZ, J . A l l a n a n d James L. SCHARDEIN.  1964.  Histochemical  studies o f four c y c l o p h y l l i d e a n cestodes.  J. Parasit.,  50:271-277. YAMAO, Y.  1952. H i s t o c h e m i c a l VII.  s t u d i e s on  endoparasites.  D i s t r i b u t i o n o f the glycero-mono-phosphatases  i n the t i s s u e s o f the cestodes Anoplocephala  p e r f o l i a t a , A. magna, M o n i e z i a b e n e d e n i , M. and T a e n i a t a e n i a e f o r m i s . Tokyo,  61:254-260.  expansa  Z o o l o g i c a l Magazine,  APPENDIX A:  O u t l i n e o f the l i f e  be  shed by  cycle of  Schistocephalus  solidus.  1.  A b o u t 20,000 eggs may  2.  Egg w i t h d e v e l o p i n g  3.  F r e e swimming c o r a c i d i u m w i t h i n n e r o n c o s p h e r e . w i t h i n 2-4 weeks a f t e r eggs a r e s h e d .  4.  C y c l o p s i s i n f e c t e d b y the c o r a c i d i u m . The c i l i a t e d l a y e r o f t h e c o r a c i d i u m i s l o s t and a p r o c e r c o i d d e v e l o p s . One h a t c h o f e g g s may i n f e c t C y c l o p s a t i n t e r v a l s o v e r a p e r i o d o f 5-6 m o n t h s . The i n f e c t i v e p r o c e r c o i d f o r m i s r e a c h e d i n a b o u t 3 o r 4 weeks b u t may s u r v i v e i n C y c l o p s f o r a t l e a s t 3 months. a. P o s t e r i o r region of Cyclops i n f e c t e d w i t h p r o c e r c o i d s , b.-f. Stages i n the development of the p r o c e r c o i d .  5.  S t i c k l e b a c k s are i n f e c t e d a f t e r i n g e s t i n g Cyclops. The c e r c o m e r i s l o s t and t h e p l e r o c e r c o i d d e v e l o p s . The p l e r o c e r c o i d becomes i n f e c t i v e a t a w e i g h t o f 10-15 mg b u t may a t t a i n a w e i g h t o f more t h a n 500 mg. a.-c. V a r i o u s s i z e d p l e r o c e r c o i d s s h o w i n g d e v e l o p m e n t o f g e n i t a l i a and proglottids.  6.  G e n i t a l i a d i f f e r e n t i a t e and e g g s f o r m w i t h i n 2-4 i s i n g e s t e d by the b i r d h o s t .  coracidium  an a d u l t worm.  n e a r l y r e a d y t o emerge. The  coracidium  days a f t e r the  may  emerge  stickleback  74  A P P E N D I X B:  Diagram o f t h e s t r u c t u r e s and r e g i o n s of solidus discussed i n the t e x t .  superficial cuticle  cuticular  cortical  medullary  Schistocephalus  parenchyma  parenchyma  The d o t t e d l i n e i n d i c a t e s t h e a p p r o x i m a t e l o c a t i o n o f t h e a l c i a n blue positive c u t i c u l a r layer.  

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