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UBC Theses and Dissertations

Relationships between seasonal biochemical changes and the reproductive cycle of the intertidal gastropod… Lambert, Philip 1970

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RELATIONSHIPS BETWEEN SEASONAL BIOCHEMICAL CHANGES AND THE REPRODUCTIVE CYCLE OF THE INTERTIDAL GASTROPOD THAIS LAMELLOSA GMELIN  (GASTROPODA, PROSOBRANCHIA)  .  by  PHILIP LAMBERT  B.Sc,  U n i v e r s i t y o f V i c t o r i a , 1967  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE  REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  i n t h e Department of Zoology  We accept t h i s t h e s i s as conforming required  THE  t o the  standard  UNIVERSITY OF BRITISH COLUMBIA June, 1970  In p r e s e n t i n g t h i s t h e s i s  in p a r t i a l  f u l f i l m e n t o f the requirements  an advanced degree at the U n i v e r s i t y of B r i t i s h C o l u m b i a , I agree the L i b r a r y  s h a l l make i t f r e e l y  available for  I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e  r e f e r e n c e and copying o f t h i s  It  i s understood that c o p y i n g o r  thesis  permission.  Department  of  The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada  Date  JULS  3L  /<?70  or  publication  o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my written  that  study.  f o r s c h o l a r l y purposes may be g r a n t e d by the Head o f my Department by h i s r e p r e s e n t a t i v e s .  for  i ABSTRACT  The  s e a s o n a l v a r i a t i o n i n the major b i o c h e m i c a l c o n s t i t u e n t s o f  T. l a m e l l o s a Gmelin have been s t u d i e d i n r e l a t i o n t o t h e r e p r o d u c t i v e cycle.  D i g e s t i v e g l a n d , f o o t muscle and  p r o t e i n , glycogen,  lipid  and  gonad were a n a l y s e d f o r  ash over a p e r i o d o f one y e a r .  In  a d d i t i o n t o biochemical analyses, h i s t o l o g i c a l sections of d i g e s t i v e gland and  gonad were made throughout  t h e same p e r i o d .  data s u p p l i e d i n f o r m a t i o n on f e e d i n g and  Histological  gamete m a t u r a t i o n .  major p e r i o d s o f f e e d i n g a c t i v i t y o c c u r r e d i n A p r i l and Garnetogenesis i n March.  Two  August.  began i n l a t e summer and t h e peak spawning p e r i o d  was  Glycogen i s a t a maximum i n t h e d i g e s t i v e gland a t times  of maximum f e e d i n g , but food i s s t o r e d i n the d i g e s t i v e gland i n t h e form o f l i p i d . winter.  Stored l i p i d  i s u t i l i z e d by the animal d u r i n g t h e  Glycogen i s a t a low l e v e l i n a l l t i s s u e s and  be used p r i m a r i l y f o r l i p i d not s t o r e e i t h e r l i p i d  i s maintained  The  f o o t muscle does  o r glycogen t o any a p p r e c i a b l e e x t e n t .  Under normal f i e l d gland index decreases  and y o l k s y n t h e s i s .  appears t o  c o n d i t i o n s during the winter, the d i g e s t i v e  as r e s e r v e s are u t i l i z e d , w h i l e t h e gonad  u n t i l spawning.  Animals which are maintained  size  through  t h e same p e r i o d under a r t i f i c i a l summer c o n d i t i o n s , show no l o s s i n t h e d i g e s t i v e gland index, but a decrease  i n t h e s i z e o f t h e gonad.  None o f t h e oogonia  r e a c h m a t u r i t y and t h e mature oocytes  The  r e s o r b more m a t e r i a l from t h e gonad t h a n f e d  s t a r v e d animals  animals.  The p o s s i b l e r o l e o f e n v i r o n m e n t a l  reproduction i s discussed.  are  resorbed.  factors in controlling  ii TABLE OF CONTENTS PAGE 1  INTRODUCTION MATERIAL AND METHODS C o l l e c t i n g Area C o l l e c t i o n o f Samples Histology Chemical A n a l y s e s Lipid Polysaccharide Protein Controlled Conditions S t a t i s t i c a l Methods  5 5 5 7 9 9 10 12 13 15  RESULTS F i e l d Observations E n v i r o n m e n t a l parameters Reproductive a c t i v i t i e s I n t e r n a l Morphology Gross anatomy H i s t o l o g i c a l observations D e t e r m i n a t i o n o f r e p r o d u c t i v e c y c l e by oocyte Parasites H i s t o l o g y o f d i g e s t i v e gland Per Cent Dry Weight Gonad Index D i g e s t i v e Gland Index Constituent Levels D i g e s t i v e gland Protein levels Glycogen and ash l e v e l s Experimental Results  16 16 16 18 18 18 21 21 24 24 26 26 29 29 29 32 32 35  diameter  DISCUSSION R e p r o d u c t i o n and Development Seasonal B i o c h e m i c a l Changes B i o c h e m i c a l c o n s t i t u e n t s o f oocytes Feeding E x p l a n a t i o n o f terms C o n s t i t u e n t s o f d i g e s t i v e gland Glycogen i n t h e d i g e s t i v e gland Role o f glycogen i n r e p r o d u c t i o n C o n s t i t u e n t s o f t h e ovary P o l y s a c c h a r i d e as a s t o r a g e product L i p i d l e v e l s i n T. l a m e l l o s a C o n s t i t u e n t s o f f o o t muscle C o n c l u s i o n s about n u t r i e n t t r a n s f e r C o n c l u s i o n s from e x p e r i m e n t a l o b s e r v a t i o n s E f f e c t o f E n v i r o n m e n t a l F a c t o r s on Gonad Development Lack o f food Temperature  42 42 46 46 47 49 49 51 52 54 55 56 57 58 59 61 61 62  iii  Day l e n g t h Salinity Suggestions f o r F u r t h e r Study  "  PAGE 63 64 64  SUMMARY  66  LITERATURE CITED  68  Iv < LIST OF TABLES PAGE  TABLE 1  Subjective c l a s s i f i c a t i o n  2  S u b j e c t i v e c l a s s i f i c a t i o n o f numbers o f granules s t o r a g e c e l l s o f t h e d i g e s t i v e gland.  3  Summary o f data f o r two e x p e r i m e n t a l groups o f T. l a m e l l o s a m a i n t a i n e d i n summer c o n d i t i o n s from August 31, 1968 t o J a n u a r y 6, 1969. Data f o r females o n l y . Numbers i n b r a c k e t s a r e 1 S.E. c^ = f i e l d = field  sample a t s t a r t  o f d i g e s t i v e gland  = f e d experimental  s  = starved experimental  22  i n the 25  o f experiment (Aug. 31).  sample a t end o f experiment ( J a n .  f  colour.  6).  group sampled a t c o n c l u s i o n , group sampled a t c o n c l u s i o n .  38  V LIST OF FIGURES FIGURE  PAGE  1  Map  2  Mean monthly s a l i n i t y two f e e t below s u r f a c e a t Brockton P o i n t . V e r t i c a l l i n e s i n d i c a t e t h e range. The f i r s t and l a s t p o i n t s do not r e p r e s e n t complete months.  14  T o t a l p r e c i p i t a t i o n f o r each 15 day p e r i o d at the P o r t M e t e o r o l o g i c a l O f f i c e , Vancouver. Calculated from t a b l e s i n the Annual M e t e o r o l o g i c a l Summary (Canada D.O.T. M e t e o r o l o g i c a l Branch, 1969, 1970).  17  Mean monthly water temperature two f e e t below s u r f a c e at Brockton P o i n t . V e r t i c a l l i n e s i n d i c a t e t h e range.  19  The p o i n t s r e p r e s e n t t h e average d a y - l i g h t hours per day f o r each month, c a l c u l a t e d from t a b l e s i n N o r t h c o t t (1968, 1969).  20  Oocyte diameter frequency polygons f o r each monthly c o l l e c t i o n o f T. l a m e l l o s a . Each polygon r e p r e s e n t s a t o t a l o f 125 oocytes and t h e p o i n t s i n d i c a t e t h e per cent o f t h e t o t a l i n each s i z e c l a s s . The a b s c i s s a i s i n per cent u n i t s .  23  Per c e n t o f dry m a t e r i a l i n a u n i t weight o f f r e s h t i s s u e o f T. l a m e l l o s a . The v e r t i c a l l i n e s i n d i c a t e + 1 S.E. Data f o r females o n l y .  27  Gonad i n d i c e s o f male and female T. l a m e l l o s a as a f u n c t i o n o f month o f year. Closed c i r c l e s ( • ) r e p r e s e n t t h e mean ovary index and open c i r c l e s ( o ) r e p r e s e n t i n d i v i d u a l males. V e r t i c a l l i n e s i n d i c a t e + 1 S.E.  28  D i g e s t i v e gland i n d i c e s o f male and female T. l a m e l l o s a as a f u n c t i o n o f month o f y e a r . Closed c i r c l e s ( • ) r e p r e s e n t t h e mean o f 8 females and t h e open c i r c l e s ( o ) r e p r e s e n t i n d i v i d u a l males. V e r t i c a l l i n e s i n d i c a t e + 1 S.E.  30  L e v e l s o f p r o t e i n , l i p i d and glycogen i n t h e d i g e s t i v e gland o f T. l a m e l l o s a as a f u n c t i o n o f month o f t h e year. Each p o i n t r e p r e s e n t s t h e per cent o f a c o n s t i t u e n t i n a u n i t weight o f dry t i s s u e . C l o s e d symbols r e p r e s e n t female means and open symbols r e p r e s e n t i n d i v i d u a l males. V e r t i c a l l i n e s i n d i c a t e + !• S.E.  31  3  4  5  6  7  8  9  10  o f r e s e a r c h area showing main sources  of run-off.  6  vi FIGURE 11  12  13  14  15  PAGE L e v e l s o f p r o t e i n , l i p i d and glycogen i n t h e f o o t muscle o f T. l a m e l l o s a as a f u n c t i o n o f month o f year. Each p o i n t r e p r e s e n t s t h e p e r cent o f a c o n s t i t u e n t i n a u n i t weight o f dry t i s s u e . Closed symbols r e p r e s e n t female means and open symbols r e p r e s e n t i n d i v i d u a l males. V e r t i c a l l i n e s i n d i c a t e + 1 S.E.  33  L e v e l s o f p r o t e i n , l i p i d and.glycogen i n t h e gonad o f T. l a m e l l o s a as a f u n c t i o n o f month o f year. Each p o i n t r e p r e s e n t s t h e p e r cent o f a c o n s t i t u e n t i n a u n i t weight o f dry t i s s u e . C l o s e d symbols r e p r e s e n t female means and open symbols r e p r e s e n t i n d i v i d u a l males. V e r t i c a l l i n e s i n d i c a t e + 1 S.E.  34  Per cent ash p e r u n i t weight o f dry t i s s u e i n t h r e e t i s s u e s o f t h e body. The data f o r d i g e s t i v e gland and f o o t muscle are means o f pooled male and female tissue.  36  Histograms r e p r e s e n t t h e amount o f dry t i s s u e accounted f o r by t h e p r o t e i n , l i p i d and glycogen d e t e r m i n a t i o n s . Data f o r ash i s a v a i l a b l e f o r o n l y t h r e e o f t h e c o l l e c t ions.  37  Oocyte diameter frequency polygons f o r two e x p e r i m e n t a l groups. E x p l a n a t i o n as i n F i g u r e 6. Animals k e p t under summer c o n d i t i o n s from August 31, 1968 u n t i l January 6, 1969.  40  vii  LIST OF  PLATES  PLATE 1  PAGE C r o s s - s e c t i o n o f d i g e s t i v e gland i l l u s t r a t i n g t h e s u b j e c t i v e c l a s s i f i c a t i o n o f granule c o n t e n t . 1 = lumen, t = t u b u l e , g = s t o r a g e g r a n u l e s . A.  tubules f u l l o f granules  B.  tubules h a l f f u l l  C.  t u b u l e s empty (3400 X)  (2600 X)  (3400 X) 25A  ACKNOWLEDGMENTS  I wish t o express  g r a t i t u d e t o my s u p e r v i s o r Dr. P.A. Dehnel  f o r h i s a s s i s t a n c e and c o n s t r u c t i v e c r i t i c i s m d u r i n g t h i s I  study.  would a l s o l i k e t o thank Dr. M.J. Smith f o r h i s a s s i s t a n c e and  encouragement d u r i n g t h e i n i t i a l s t a g e s o f t h e r e s e a r c h , C. Rauchert who a d v i s e d me on some o f t h e b i o c h e m i c a l t e c h n i q u e s , D. Kramer f o r h i s a d v i c e and a s s i s t a n c e w i t h t h e photography, Dr. H. Nordan f o r making a v a i l a b l e f a c i l i t i e s f e l l o w graduate mention.  f o r ash d e t e r m i n a t i o n , and my  s t u d e n t s f o r a s s i s t i n g me i n ways t o o numerous t o  1 INTRODUCTION  Reproductive  c y c l e s o f v a r i o u s marine i n v e r t e b r a t e s based  on  h i s t o l o g i c a l i n v e s t i g a t i o n s o r gonad i n d i c e s are demonstrated i n the l i t e r a t u r e .  Giese (1959) reviewed  t h e work up t o t h a t time  and s i n c e t h e n f u r t h e r s p e c i e s have been i n v e s t i g a t e d : K a t h e r i n a t u n i c a t a and M o p a l i a h i n d s i i ( G i e s e , Tucker,  The c h i t o n s , and B o o l o o t i a n ,  1959b) and C r y p t o c h i t o n s t e l l e r i (Lawrence, Lawrence, and 1965) ; t h e abalone,  Giese,  H a l i o t i s c r a c h e r o d i i (Webber and G i e s e , 1969);  t h e l i m p e t s , Acmaea m i t r a ( F r i t c h m a n , 1961), Acmaea l i m a t u l a (Seapy, 1966) , and F i s s u r e l l a b a r b a d e n s i s  (Ward, 1966); t h e  littorines,  L i t t o r i n a p i n t a d o , L. p i c t a , and L. s c a b r a ( S t r u h s a k e r , 1966); t h e b i v a l v e s , C r a s s o s t r e a v i r g i n i c a (Sakuda, 1966), A e q u i p e c t e n  irradians  ( S a s t r y , 1966), S p i s u l a s o l i d i s s m a (Ropes, 1968), M y t i l u s e d u l i s p l a n u l a t u s , Xenostrobus  pulex, S e p t i f e r b i l o c u l a r i s ,  Brachidontes  v a r i a b i l i s , and Amygdalum glaberrimum ( W i l s o n and Hodgkin, 1967); t h e a s t e r o i d s , Odontaster  v a l i d u s ( P e a r s e , 1965)  and P i s a s t e r  ochraceus  (Mauzey, 1966); t h e o p h i u r o i d , Gorgonocephalus c a r y i ( P a t e n t , 1969). B i o c h e m i c a l changes a s s o c i a t e d w i t h t h e r e p r o d u c t i v e c y c l e have not been e x t e n s i v e l y s t u d i e d ( G i e s e e t a l . 1959a; Giese and 1962;  Barnes,  Barnes and F i n l a y s o n , 1963;  G i e s e and H a r t , 1967;  Blackmore, 1969).  A n s e l l and Lander,  o r spawning p o t e n t i a l , t h e l a t t e r b e i n g determined Few  1967;  F o r t h e most p a r t , t h e  r e p r o d u c t i v e c y c l e s i n t h e s e s t u d i e s were determined  ing stimuli.  Araki,  by gonad  indices  by standard spawn-  s t u d i e s u t i l i z e d t h e more p r e c i s e method o f measur-  i n g oocyte s i z e s i n h i s t o l o g i c a l s e c t i o n s t o determine the reproductive cycle.  The  t h e stage o f  data o b t a i n e d i n t h e s e s t u d i e s p r o v i d e  2 a more complete  p i c t u r e o f t h e i n t e r n a l changes,  and a l l o w s t h e i n -  v e s t i g a t o r t o p o s t u l a t e t h e r e l a t i o n s h i p between n u t r i t i o n and t h e reproductive cycle.  F o r example, Giese and Hart (1967) found s e a s o n a l  changes i n t h e gonad and d i g e s t i v e gland i n d i c e s o f K a t h e r i n a t u n i c a t a throughout t h e y e a r . remained for  A n a l y s e s showed t h a t most o f t h e body components  similar i n t h e i r protein, l i p i d  t h e ovary and t e s t i s .  and t o t a l c a r b o h y d r a t e except  The l a t t e r organs showed marked changes i n  p r o t e i n and c a r b o h y d r a t e l e v e l ,  and t h e ovary showed a change i n l i p i d  l e v e l which reached a maximum a t t h e h e i g h t o f t h e b r e e d i n g season. The c a r b o h y d r a t e l e v e l i n t e s t i s and ovary was maximal d u r i n g t h e time o f minimum gonad s i z e and minimal a t t h e peak o f b r e e d i n g .  From t h e s e  d a t a , t h e y suggest t h a t as t h e ovary d e v e l o p s , c a r b o h y d r a t e i s u t i l i z e d by t h e o o c y t e s .  They a l s o suggest t h a t c a r b o h y d r a t e i s c o n v e r t e d t o  p r o t e i n i n the t e s t i s , while l i p i d  remains  fairly  suggested t h a t c a r b o h y d r a t e i s c o n v e r t e d t o l i p i d ovary.  constant.  It is  and p r o t e i n i n t h e  Whether t h a t i s i n f a c t what o c c u r s would r e q u i r e more s p e c i f i c  i n v e s t i g a t i o n , perhaps  involving radioactive tracers.  However, b i o -  c h e m i c a l s t u d i e s o f t h i s t y p e can a t l e a s t p r o v i d e a b a s i s f o r e x p l a i n ing  t h e mechanisms i n v o l v e d i n t h e t i m i n g and c o n t r o l o f r e p r o d u c t i o n . E a r l y work o f t h i s t y p e was  done on c o m m e r c i a l l y important b i v a l v e s  such as t h e o y s t e r s , O s t r e a g i g a s and 0. e d u l i s ( M i t c h e l l , 1915-16; R u s s e l l , 1923;  Masumoto, Masumoto, and H i b i n o , 1934;  Hatanaka,  1940)  and t h e mussel, M y t i l u s e d u l i s ( D a n i e l , 1921), w h i l e more r e c e n t l y , b i o c h e m i c a l work has been performed and c h i t o n s . this field  on o t h e r groups  such as  echinoderms  G i e s e and h i s c o l l e a g u e s have c o n t r i b u t e d many papers t o  of invertebrate physiology.  There i s an obvious l a c k o f  3  biochemical two  i n f o r m a t i o n on marine prosobranch gastropods except f o r  r e c e n t papers on P a t e l l a v u l g a t a (Blackmore, 1969) and on H a l i o t i s  c r a c h e r o d i i (Webber and Giese, 1969). t i d a l gastropod,  Consequently, t h e marine  T h a i s l a m e l l o s a Gmelin, a common s p e c i e s i n t h e area  o f Vancouver, B r i t i s h Columbia, was chosen f o r t h e p r e s e n t study  inter-  study.  A  o f t h i s t y p e would p r o v i d e b a s i c i n f o r m a t i o n on t h e changes i n  biochemical  c o n s t i t u e n t s and t h e r e p r o d u c t i v e c y c l e o f T h a i s  which might be important  lamellosa,  t o further e c o l o g i c a l or p h y s i o l o g i c a l i n -  vestigation. Some i n f o r m a t i o n has been p u b l i s h e d l a m e l l o s a from f i e l d capsule  observations  on t h e r e p r o d u c t i o n o f T.  o f breeding  d e p o s i t i o n ( G r i f f i t h , 1967).  aggregations  and egg-  Emlen (1966) d i d an e c o l o g i c a l  study o f T. l a m e l l o s a from t h e p o i n t o f view o f t i m e , energy and r i s k and,, p r o v i d e d  i n f o r m a t i o n on times o f c a p s u l e l a y i n g , l e n g t h o f b r e e d -  i n g season, and developmental time., i n a p o p u l a t i o n from P o r t Townsend, Washington.  Chapman and Banner (1949) r e p o r t t h a t t h e n a t i v e  drill,  T. l a m e l l o s a , l a y s eggs i n Puget Sound between March and J u n e . a l s o performed some simple T. l a m e l l o s a . salinities  They  experiments on t h e s a l i n i t y t o l e r a n c e o f  A f t e r 11 days a t 15.2°/oo, 3 out o f 10 d i e d ,  above t h i s had no o b s e r v a b l e  effect.  while  As t h e s a l i n i t y  dropped below about 15°/°° "the s n a i l s tended t o c l o s e up and remain i n a c t i v e ; hence, t h e s a l i n i t y i n d i r e c t l y a f f e c t e d f e e d i n g . o f T. l a m e l l o s a appears t o be mussels and b a r n a c l e s  The d i e t  with a p r e f e r e n c e  f o r t h e l a t t e r ( K i n c a i d , 1957). D a l l (1915) reviewed t h e taxonomic h i s t o r y o f T h a i s  (Nucella)  l a m e l l o s a Gmelin, and d e s c r i b e d f i v e v a r i e t i e s w i t h type l o c a t i o n s a t  4 f o u r p l a c e s on t h e P a c i f i c c o a s t .  K i n c a i d (1957) p r e s e n t e d many  i l l u s t r a t i o n s o f s h e l l v a r i a t i o n s from d i f f e r e n t l o c a t i o n s on t h e P a c i f i c coast.  F o r t h e purposes  o f t h i s study, animals were c o l l e c t e d  from one l o c a t i o n and i d e n t i f i e d as T h a i s l a m e l l o s a from t h e d e s c r i p t i o n of  Griffith  (1967), and no c o n s i d e r a t i o n was given t o t h e d i f f e r e n t  varieties. In  t h i s paper, t h e b i o c h e m i c a l c o n s t i t u e n t s :  lipid,  carbohydrate,  p r o t e i n , and ash; i n gonad, d i g e s t i v e g l a n d , and muscle t i s s u e , were determined  f o r monthly samples and, t h e r e p r o d u c t i v e c y c l e was d e t e r -  mined from h i s t o l o g i c a l data and gonad i n d i c e s . meters o f temperature  Environmental  para-  and s a l i n i t y were r e c o r d e d c o n c u r r e n t l y w i t h  f i e l d c o l l e c t i o n s and, a l i m i t e d amount o f e x p e r i m e n t a t i o n was p e r formed i n t h e l a b o r a t o r y under c o n t r o l l e d c o n d i t i o n s .  T h i s study was  designed t o p r o v i d e some b a s i c i n f o r m a t i o n which w i l l s e r v e as t h e b a s i s f o r f u r t h e r e x p e r i m e n t a t i o n on T h a i s l a m e l l o s a .  5 MATERIAL AND METHODS  C o l l e c t i n g Area A sample o f about 20 T h a i s l a m e l l o s a was c o l l e c t e d each month a t B r o c k t o n P o i n t , S t a n l e y Park, Vancouver,  B r i t i s h Columbia,  and t i s s u e was o b t a i n e d from 8 females and 2 males.  Animals were  c o l l e c t e d between t h e 0 and 3 f o o t t i d e l e v e l s , t h e maximum h e i g h t a t t h i s l o c a t i o n b e i n g about 15 f e e t .  ( F i g . 1)  tidal  Only t h o s e animals  l o n g e r t h a n 4 cm. were chosen, as s m a l l e r animals d i d n o t y i e l d s u f f i c i e n t t i s s u e f o r i n d i v i d u a l chemical analyses. s e c t i o n , animals were k e p t i n a e r a t e d sea-water temperatures, 15°C i n summer and 8°C i n w i n t e r .  Prior to dis-  approximating These  field  temperatures  were c o n t r o l l e d by t h e c o l d t a p water on a water t a b l e and were s u b j e c t t o t h e changes i n t h e water s u p p l y . tained  The animals were main-  i n t h i s way f o r a t l e a s t 3 days t o a l l o w t h e gut t o empty.  Water temperatures were r e c o r d e d a t a depth o f 2 f e e t w i t h a Temps c r i b e c o n t i n u o u s temperature r e c o r d e r (GBI S c i e n t i f i c ) o f f t h e Texaco O i l barge a d j a c e n t t o t h e c o l l e c t i n g a r e a .  A water sample was c o l -  l e c t e d once a week and s a l i n i t y was determined by s t a n d a r d t i t r a t i o n methods. C o l l e c t i o n o f Samples The s o f t p a r t s were removed by c r a c k i n g t h e s h e l l open.  External  water on t h e whole animal was b l o t t e d w i t h absorbant t i s s u e and t h e t o t a l s o f t p a r t s , i n c l u d i n g t h e operculum, l o a d i n g b a l a n c e t o + 0.01 grams.  were weighed on a t o p -  A p o r t i o n of the gonad-digestive  gland complex was removed and f i x e d i n G i l s o n ' s F l u i d K o z l o f f , 1964) f o r l a t e r s e c t i o n i n g .  ( G a l i g h e r and  The gonad, d i g e s t i v e gland and  6  FIGURE 1 Map  o f r e s e a r c h a r e a showing main sources  of run-off.  7 f o o t muscle were d i s s e c t e d o f f , t r a n s f e r r e d t o p i e c e s o f P a r a f i l m and weighed t o + 0.1 mg.  The t i s s u e  was d r i e d i n a vacuum  dessicator  over D r i e r i t e f o r 48 hours which removed 98-99% o f t h e water.  From  t h i s procedure, % d r y weight,  index  were o b t a i n e d . t o wet weight  gonad index, and d i g e s t i v e gland  The p e r cent dry weight  i s t h e r a t i o o f d r y weight  and i s a measure o f t h e t o t a l s o l i d s  i n t h e wet  tissue.  S i n c e p a r t o f t h e complex had been removed f o r f i x a t i o n , a t r u e component index c o u l d n o t be o b t a i n e d ; however, as near as p o s s i b l e , a p r o p o r t i o n a l amount was removed from each animal.  The component  o b t a i n e d t h e n , was a r e l a t i v e r a t h e r t h a n an a b s o l u t e v a l u e .  index  There  appears t o be some c o n f u s i o n i n t h e l i t e r a t u r e r e g a r d i n g component index.  Some authors c a l c u l a t e t h e index from t h e r a t i o o f component  volume t o t o t a l wet weight times 100 ( G i e s e , e t a l . , 1959a; Giese, e t a l . , 1959b; Giese and A r a k i , 1962), w h i l e i n more r e c e n t papers t h e r a t i o o f component weight t o t o t a l wet weight was used ( G i e s e , 1967; Giese and H a r t , 1967; S a s t r y , 1968; Webber and G i e s e , 1969). The l a t t e r method was used  in this  study.  Histology Several f i x a t i v e s  were t e s t e d , i n c l u d i n g Baker's,  Bouin's, and G i l s o n ' s f i x a t i v e s . satisfactory tissues.  results  Carnoy's,  The l a s t was found t o g i v e t h e most  and was employed as t h e f i x a t i v e f o r a l l t h e  L a t e r i n t h e course o f t h e study, P i c r o - N i t r i c  fixative  was found t o g i v e b e t t e r f i x a t i o n o f y o l k b u t t o m a i n t a i n u n i f o r m i t y o f s h r i n k a g e o f oocytes G i l s o n s F l u i d was used throughout T  t h e study.  The method o f oocyte measurement w i l l be d e s c r i b e d l a t e r . A segment from t h e d i s t a l end o f t h e g o n a d - d i g e s t i v e gland complex  8 was f i x e d i n G i l s o n ' s F l u i d over n i g h t .  S e c t i o n i n g o f t h e whole gonad  i n d i c a t e d t h a t development o f t h e oocytes was s y n c h r o n i z e d throughout. The t i s s u e was t h e n washed f o r s e v e r a l hours remove excess f i x a t i v e .  i n r u n n i n g tap-water t o  The t i s s u e was dehydrated up t o 70% EtOH  where i t c o u l d be s t o r e d u n t i l p r o c e s s e d .  From t h i s p o i n t t h e t i s s u e  was dehydrated w i t h EtOH, c l e a r e d i n benzene and embedded i n Tissuemat (m.p. 56.7°C).  The m a j o r i t y o f t h e s e c t i o n s were c u t a t 8 microns b u t  some ranged from 5-10 microns depending  on f a c t o r s such as s t a t i c  e l e c t r i c i t y and sharpness o f b l a d e a t t i m e o f s e c t i o n i n g , and f i x a t i o n o f t h e specimen.  Masson's Trichrome s t a i n ( P a n t i n , 1964) was employed  and gave b l a c k n u c l e i , green mucus and c o n n e c t i v e t i s s u e , p i n k e p i t h e l i u m , and orange y o l k g r a n u l e s . s e c t i o n s were exposed  D u r i n g t h e s t a i n i n g procedure t h e  t o Lugol's I o d i n e i n 70% EtOH which s e r v e d t o  remove c r y s t a l s o f m e r c u r i c c h l o r i d e r e s u l t i n g from t h e f i x a t i v e . The  gonad s e c t i o n s were a n a l y s e d by t h e method o f Pearse  (1965).  With an e y e p i e c e s c a l e c a l i b r a t e d i n microns, two measurements o f oocyte diameter were made a t r i g h t a n g l e s t o each o t h e r , t h e s e u s u a l l y b e i n g t h e l o n g e s t and s h o r t e s t axes.  From t h e s e two p e r p e n d i c u l a r  measurements t h e mean diameter o f each oocyte was c a l c u l a t e d .  These  d a t a were o b t a i n e d f o r 25 randomly s e l e c t e d oocytes i n each o f 5 females. Only t h o s e oocytes which were s e c t i o n e d through t h e n u c l e u s were measured.  Some o f t h e l a r g e mature oocytes p r e s e n t e d problems i n measure-  ment s i n c e t h e n u c l e u s was p y k n o t i c and d i f f i c u l t t o see among t h e l a r g e y o l k g r a n u l e s ; f u r t h e r m o r e , t h e membranes o f t h e mature oocytes were d i f f i c u l t t o d i s c e r n so t h e boundaries o f t e n had t o be e s t i m a t e d  9 u s i n g c l u e s from t h e shape o f t h e s u r r o u n d i n g t i s s u e and n e i g h b o u r i n g oocytes. graph or  A l l t h e measurements were arranged  i n t o s i z e c l a s s e s and a  o f s i z e d i s t r i b u t i o n was drawn ( F i g . 6 ) .  polygons  i n d i c a t e the per cent frequency  each monthly c o l l e c t i o n throughout The  The i n d i v i d u a l graphs  i n each s i z e c l a s s f o r  the year.  s e c t i o n s o f d i g e s t i v e gland were a n a l y s e d f o r t h e i r  granule content.  storage  The columnar c e l l s o f t h e d i g e s t i v e gland t u b u l e s  were s u b j e c t i v e l y c l a s s i f i e d  as b e i n g f u l l o f g r a n u l e s , h a l f f u l l , o r  empty ( T a b l e 2, P l a t e 1 ) . Chemical  Analyses  Chemical performed  analyses f o r l i p i d ,  p r o t e i n , and p o l y s a c c h a r i d e were  on t h e d r y t i s s u e from i n d i v i d u a l animals r a t h e r t h a n on  pooled t i s s u e s from s e v e r a l animals.  I n d i v i d u a l determinations  pro-  v i d e d i n f o r m a t i o n on t h e amount o f v a r i a t i o n i n t h e p o p u l a t i o n and t h e d i f f e r e n c e s between males and females, as w e l l as I n d i v i d u a l data which c o u l d be c o r r e l a t e d w i t h t h e h i s t o l o g i c a l r e s u l t s , of one  c o l o u r , and p e r cent water i n t h e t i s s u e .  observations  For p r a c t i c a l  d e t e r m i n a t i o n o f each component was performed  reasons,  i n each t i s s u e  sample,  as opposed t o p o o l i n g t h e t i s s u e s and d o i n g s e v e r a l r e p l i c a t e s . Lipid:  Lipid  and p o l y s a c c h a r i d e d e t e r m i n a t i o n s were performed  t h e same p o r t i o n o f t i s s u e . method based of  on  T o t a l l i p i d v a l u e s were o b t a i n e d by a  on t h a t o f Sperry and Brand (1955).  Approximately  20 mg.  d r y t i s s u e were homogenized i n 2 ml. o f a chloroform-methanol  sol-  u t i o n (2:1) w i t h a t i s s u e g r i n d e r (Pyrex Brand) and t r a n s f e r r e d t o a c e n t r i f u g e tube.  The g r i n d e r was r i n s e d s e v e r a l times and t h e washings  were added t o t h e sample.  The homogenate was c e n t r i f u g e d and t h e  10 supernatant c o n t a i n i n g the l i p i d The r e m a i n i n g p r e c i p i t a t e was  retained f o r polysaccharide  which w i l l be d e s c r i b e d l a t e r . s o l u t i o n o f 1.6%  CaCl  w  a  2  s  was t r a n s f e r r e d t o a screw-cap  To t h e l i p i d  analysis,  e x t r a c t , 5 ml. o f a wash  added which, a f t e r s h a k i n g f o r 5 min.,  caused t h e s e p a r a t i o n o f t h e l i p i d upper l a y e r was  vial.  layer.  A f t e r c e n t r i f u g a t i o n , the  removed and d i s c a r d e d and about 2 ml. o f a second  wash s o l u t i o n , chloroform-methanol (2:1) p l u s 2% C a C l ^ ( S p e r r y and Brand, 1955), was lipid  l a y e r e d c a r e f u l l y on t h e s u r f a c e t o remove any non-  i m p u r i t i e s remaining at the surface.  T h i s wash l a y e r was  removed  w i t h a P a s t e u r p i p e t t e a f t e r c e n t r i f u g a t i o n , and t h e r e m a i n i n g l i p i d was  evaporated t o dryness a t 60°C w i t h a stream o f n i t r o g e n .  This  l a t t e r procedure was t o reduce t h e p o s s i b i l i t y o f o x i d i z i n g t h e The l i p i d  r e m a i n i n g was  t h e n d i s s o l v e d w i t h about 1 ml. o f e t h e r and  t r a n s f e r r e d t o a pre-weighed was  tube  added t o t h e  When d r y , t h e weight o f the e x t r a c t e d  determined and t h e t o t a l l i p i d  c u l a t e d as a p e r c e n t a g e .  lipid  / u n i t d r y weight o f t i s s u e was  The percentage l i p i d  weight w i l l be r e f e r r e d t o as t h e l i p i d the  The  The sponge a l l o w e d f o r f a s t e r e v a p o r a t i o n and a l s o prevented  s p i l l a g e o f t h e sample. was  p i e c e o f sponge on a p l a n c h e t .  r i n s e d a second time w i t h 1 ml. o f e t h e r and was  planchet.  lipid.  cal-  p r e s e n t p e r u n i t dry  l e v e l i n t h i s paper,  following  t e r m i n o l o g y i n t r o d u c e d by Giese (1967). Polysaccharide:  r e s i d u e from t h e l i p i d analysis.  As mentioned  i n the l i p i d  e x t r a c t i o n was  a n a l y s i s method, t h e  used f o r t h e p o l y s a c c h a r i d e  T h i s has two advantages: ( l ) both d e t e r m i n a t i o n s can be  performed on one s m a l l amount o f t i s s u e , and (2) t h e p r i o r removal o f lipid  p r e v e n t s foaming d u r i n g t h e p o l y s a c c h a r i d e e x t r a c t i o n  (Giese,  11 1967).  The t i s s u e was,  first  o f a l l , heated with 4 ml.  o f 80% EtOH  t o p r e c i p i t a t e t h e p o l y s a c c h a r i d e and remove some s o l u b l e i m p u r i t i e s . The  s o l u t i o n was  p r e c i p i t a t e was  c e n t r i f u g e d and t h e supernatant heated  with 4 ml.  o f TCA  discarded.  The  a t 100°C t o e x t r a c t t h e  p o l y s a c c h a r i d e , t h e s u p e r n a t a n t from the e x t r a c t i o n b e i n g p l a c e d i n a 25 ml. v o l u m e t r i c f l a s k .  T h i s e x t r a c t i o n was  performed  t o ensure complete removal o f t h e p o l y s a c c h a r i d e . was  t h r e e times  A 2 ml.  aliquot  then a n a l y s e d f o r p o l y s a c c h a r i d e by t h e anthrone method o f  S e i f t e r , e t a l . (1950) with t h e m o d i f i c a t i o n s o f H e l b e r t and (1955).  The  Brown  anthrone method depends on d e h y d r a t i o n o f t h e sugar t o  a f u r f u r a l d e r i v a t i v e by t h e s u l f u r i c a c i d component which combines w i t h t h e anthrone o f anthrone  t o form a b l u e - c o l o u r e d compound.  i n 95% s u l f u r i c a c i d was  s o l u t i o n when 4 hours o l d , was l a t t e r was  A 0.16%  made up f r e s h d a i l y .  solution  The  anthrone  added t o the 2 ml. sample w h i l e t h e  i n an i c e - b a t h and b e i n g a g i t a t e d w i t h a magnetic  stirrer.  The tubes were s e a l e d w i t h g l a s s marbles and P a r a f i l m and p l a c e d i n a b o i l i n g water-bath  f o r 10 min.  (+ 2 s e e s . ) .  t a i n t h e bath at a c o n s t a n t temperature precisely for consistent results.  I t i s important t o main-  and t o time t h e i n c u b a t i o n  A f t e r 10 min.  a t 100°C t h e  were a g a i n p l a c e d i n t h e i c e - b a t h t o s t o p t h e r e a c t i o n . were then allowed t o e q u i l i b r a t e w i t h room temperature and read i n a Beckman DU millimicrons.  spectrophotometer  An a b s o r p t i o n spectrum  and t h e p o i n t o f maximum O.D.  was  verified  c o n c e n t r a t i o n o f p o l y s a c c h a r i d e was prepared  from g l u c o s e s t a n d a r d s .  was  The  tubes tubes  (about 20  a t a wavelength o f  min.)  625  run under t h e same c o n d i t i o n s as b e i n g 625 mu.  The  c a l c u l a t e d from a s t a n d a r d  The r e s u l t s were expressed  curve  as p e r  c e n t p o l y s a c c h a r i d e i n t h e dry t i s s u e and w i l l be r e f e r r e d t o as  12 polysaccharide Protein;  level. The Lowry c o l o r i m e t r i c method was  used f o r p r o t e i n  d e t e r m i n a t i o n s , bovine albumin s e r v i n g as t h e s t a n d a r d (Lowry, 1951).  Approximately  5 mg.  o f d r y t i s s u e was  dissolved  1 N. NaOH f o r s e v e r a l hours and d i l u t e d t o 25 ml. was  et a l . ,  i n 4 ml.  A 1 ml.  of  aliquot  used f o r t h e p r o t e i n d e t e r m i n a t i o n s f o l l o w i n g t h e procedure  Lowry, e t a l . (1951).  of  I n r e c e n t y e a r s t h e Lowry c o l o r i m e t r i c method  f o r d e t e r m i n i n g p r o t e i n s has come i n t o widespread use T h i s method has s e v e r a l advantages." r e a g e n t but r e q u i r e s no d i g e s t i o n .  ( G i e s e , 1967).  ( l ) I t i s as s e n s i t i v e as N e s s l e r ' s (2) I t i s 10 o r 20 t i m e s more  s e n s i t i v e t h a n t h e measurement o f t h e u l t r a v i o l e t  a b s o r p t i o n a t 280  mu.  and i s much more s p e c i f i c and much l e s s l i a b l e t o d i s t u r b a n c e by t u r bidities.  (3) I t i s s e v e r a l f o l d more s e n s i t i v e t h a n t h e n i n h y d r i n  r e a c t i o n and  i s somewhat s i m p l e r , as w e l l as much e a s i e r t o adapt t o  small scale analyses. biuret reaction. action,  (4) I t i s 100 times more s e n s i t i v e t h a n t h e  There are two major disadvantages t o t h e Lowry r e -  ( l ) The amount o f c o l o u r v a r i e s w i t h d i f f e r e n t  however, i n t h i s study t h e same t y p e o f t i s s u e was  a n a l y s e d through  t h e y e a r so o n l y t h e r e l a t i v e changes were important. i s not s t r i c t l y p r o p o r t i o n a l t o c o n c e n t r a t i o n . ficulty,  proteins;  (2) The c o l o u r  To overcome t h i s  dif-  sample c o n c e n t r a t i o n s were k e p t i n t h e range o f 0 t o 100 |j.g./ml.  where t h e s t a n d a r d c u r v e was  essentially  The 1 ml. sample a l i q u o t was carbonate-copper  linear.  allowed t o s i t w i t h 5 ml. o f a  s o l u t i o n f o r 10 min.  and t h e n t o t h i s m i x t u r e  was  added 0.5 ml. o f t h e F o l i n - C i o c a l t e a u p h e n o l reagent w h i l e t h e tube shaken. 30 min.  The c o l o u r was and was  was  allowed t o develop a t room temperature f o r  measured i n a Beckman DU spectrophotometer  a t 750  mo-.  13 An a b s o r p t i o n spectrum was o b t a i n e d t o v e r i f y 750 mu as t h e wavelength o f maximum a b s o r p t i o n .  The c o n c e n t r a t i o n was expressed as t h e p e r cent  p r o t e i n p e r u n i t d r y weight o f t i s s u e .  As w i t h l i p i d  and g l y c o g e n ,  t h i s v a l u e w i l l be r e f e r r e d t o as t h e p r o t e i n l e v e l i n t h e t i s s u e . Ash weights were determined on p o o l e d samples o f d r y t i s s u e f o r t h r e e p e r i o d s i n t h e y e a r u s i n g t h e method d e s c r i b e d o f A n a l y s i s o f t h e A.O.A.C. (1960, page 419). divided  i n O f f i c i a l Methods  The p o o l e d sample was  i n t o two a l i q u o t s and t h e mean o f t h e ash weights  calculated.  A s h i n g was done a t a temperature o f 525°C f o r about 12 hours t o c o n s t a n t weight. Controlled  Conditions  Two groups o f T h a i s l a m e l l o s a , about 20 i n each, were m a i n t a i n e d under c o n t r o l l e d "summer" c o n d i t i o n s o f s a l i n i t y a t u r e (15°C) and l i g h t  (16 hours l i g h t ;  d i s h e s s u p p l i e d w i t h a e r a t e d sea-water.  (l8.9°/oo), temper-  8 hours dark) i n p l a s t i c The s a l i n i t y o f 18.9°/op  approximated t h e low v a l u e s r e c o r d e d i n May and June s t r e n g t h sea-water was d i l u t e d w i t h g l a s s d i s t i l l e d d e s i r e d v a l u e , and was changed weekly.  ( F i g . 2).  Full  water t o t h e  The temperature o f 15°C  approximated t h e f i e l d water temperature i n t h e summer ( F i g . 4) and was r e g u l a t e d by a t h e r m o s t a t i c a l l y c o n t r o l l e d water-bath (+ 1.0°C). The l i g h t regime r e p r e s e n t e d t h e day l e n g t h i n June ( F i g . 5) and was a u t o m a t i c a l l y c o n t r o l l e d by a t i m i n g mechanism. One group o f animals was s u p p l i e d w i t h mussels and b a r n a c l e s a t a l l t i m e s , w h i l e t h e o t h e r was s t a r v e d .  Organisms  such as b a r n a c l e s  growing on t h e s h e l l s o f t h e s t a r v e d group were removed and t h e w a l l s o f t h e d i s h e s were k e p t c l e a n o f a l g a e .  A t t h e end o f 4 months t i s s u e s  FIGURE 2  Mean monthly s a l i n i t y two f e e t below t h e s u r f a c e a t Brockton Point. V e r t i c a l l i n e s i n d i c a t e t h e range. The f i r s t and l a s t p o i n t s do n o t r e p r e s e n t complete months.  1968  MONTH OF YEAR  1969  of  8 females  and  2 males were o b t a i n e d and  p o l y s a c c h a r i d e , dry weight, described previously.  analysed f o r p r o t e i n ,  lipid,  and component index u s i n g t h e methods  A l s o gonads and  d i g e s t i v e glands were s e c t i o n e d  and a n a l y s e d f o r oocyte s i z e d i s t r i b u t i o n and Data o b t a i n e d from r e g u l a r f i e l d  storage granule content.  samples a t t h e b e g i n n i n g and end  of  t h e e x p e r i m e n t a l p e r i o d r e p r e s e n t e d t h e normal changes d u r i n g t h a t time. S t a t i s t i c a l Methods Student's t - t e s t was between mean v a l u e s .  used t o determine  significant  differences  P l e v e l s o f 5% or l e s s were accepted as  signifi-  cant. Photographs were t a k e n through bright f i e l d  a Z e i s s Photo-Microscope  o p t i c s on Kodak Panatomic-X f i l m .  w i t h Masson's T r i c h r o m e .  with  S e c t i o n s were s t a i n e d  16 RESULTS  Field  Observations E n v i r o n m e n t a l parameters:  F i g u r e 2 shows t h e s a l i n i t y  over a 14 month p e r i o d from J u l y 1968 u n t i l September 1969. maximum s a l i n i t y  o f 28.5°/oo, o c c u r r e d  The  i n t h e middle o f March 1969;  t h e minimum o f 18°/oo, a t t h e end o f May and t h e b e g i n n i n g T h i s minimum corresponded t o t h e maximum d i s c h a r g e River.  changes  of July.  o f the Fraser  D u r i n g t h e w i n t e r t h e F r a s e r R i v e r d i s c h a r g e h e l d steady a t  about 25,000 c u b i c f e e t / s e c . and a t maximum d i s c h a r g e  reached 425,000  cubic feet/sec. ( P a c i f i c  The low s a l i n i t i e s  Oceanographic Group, 1951).  o c c u r r i n g i n September and November can be e x p l a i n e d by t h e r a i n f a l l data o f F i g u r e  3 (Canada D.O.T. M e t e o r o l o g i c a l  of r a i n f a l l a t the beginning  i n t h e second h a l f o f September.  salinity  i n November p r o b a b l y  S i m i l a r l y , the dip i n  r e s u l t e d from t h e r a i n f a l l peak i n e a r l y  From December t o March t h e s a l i n i t y  increased  most o f t h e p r e c i p i t a t i o n f e l l as snow i n t h e s u r r o u n d i n g and  r u n - o f f was c o n s e q u e n t l y low.  s l o w l y as mountains  From A p r i l t o J u l y t h e s a l i n i t y  dropped as snows melted and r u n - o f f i n c r e a s e d .  The main sources o f  r u n - o f f i n t h e c o l l e c t i o n area a r e t h e Squamish R i v e r , C a p i l a n o F r a s e r R i v e r , Seymour Creek, and numerous s m a l l e r creeks mountains around B u r r a r d  A peak  o f September r e s u l t e d i n a drop o f  salinity  November.  Branch, 1968).  River,  draining the  Inlet (Fig.l ) .  F i g u r e 4 shows t h e water temperature v a r i a t i o n over a p e r i o d o f 12 months from August 1968 t o J u l y 1969. 2°C  occurred  temperatures.  The minimum temperature o f  i n l a t e December as a r e s u l t o f u n s e a s o n a l l y  low a i r  The maximum o f 15.5°C was i n J u l y 1969 a t t h e end o f  FIGURE 3  T o t a l p r e c i p i t a t i o n f o r each 15 day p e r i o d a t t h e P o r t Meteorol o g i c a l O f f i c e , Vancouver. C a l c u l a t e d from t a b l e s i n t h e Annual M e t e o r o l o g i c a l Summary (Canada D.O.T. M e t e o r o l o g i c a l Branch, 1969, 1970).  Vancouver  i S B J - i  A  S  1968  O  I  3 i• i Ss I i §§i i i - s § i j j _ J §  N  D  J  E  s  F  MONTH OF YEAR  M  A  •  M  J  1969  J  A  the recording period.  A p l o t o f d a y l i g h t hours ( F i g . 5) shows t h a t  t h e minimum water temperature corresponded t o t h e s h o r t e s t day and t h e maximum water temperature lagged behind  t h e maximum  length,  day  l e n g t h by about a month. Reproductive  activities:  egg-capsules at t h e b e g i n n i n g was  T. l a m e l l o s a was  first  observed t o l a y  o f January when t h e water temperature  near a minimum and t h e s a l i n i t y was  about 25°/oo.  No  observations  were made on t h e d u r a t i o n o f c a p s u l e l a y i n g f o r t h e p o p u l a t i o n h i s t o l o g i c a l d a t a , which w i l l be d e s c r i b e d l a t e r , continues  u n t i l about A p r i l .  The  of rocks.  i n d i c a t e the l a y i n g  p o p u l a t i o n o f T. l a m e l l o s a appeared  t o move t o lower t i d a l l e v e l s d u r i n g t h e w i n t e r aggregations  but  and  e i t h e r formed  f o r r e p r o d u c t i o n or b u r i e d themselves around t h e bases  They were found o n l y i n t h e b a r n a c l e  zone and  seldom as  h i g h as t h e M y t i l u s zone, so i t appeared t h a t T. l a m e l l o s a was m o s t l y on b a r n a c l e s ; however, these  feeding  are o n l y c a s u a l o b s e r v a t i o n s made  d u r i n g c o l l e c t i o n t r i p s and r e q u i r e more documentation. I n t e r n a l Morphology Gross anatomy:  The  sexes are s e p a r a t e  i n T. l a m e l l o s a ; however,  t h e r e are no e x t e r n a l d i f f e r e n c e s except f o r t h e presence o f a p e n i s i n t h e male which can sometimes be seen d u r i n g c o p u l a t i o n . morphology o f T. l a m e l l o s a i s v i r t u a l l y i d e n t i c a l t o N u c e l l a  The lapillus,  an A t l a n t i c s p e c i e s which i s d e s c r i b e d i n d e t a i l i n F r e t t e r and (1962, pages 332-338). gland  The  gonad l i e s on one  i n t h e upper c o i l s of t h e v i s c e r a l mass.  y e l l o w and  Graham  s i d e of the d i g e s t i v e The  ovary i s u s u a l l y  g r a n u l a r i n appearance, w h i l e the t e s t i s i s more t r a n s -  l u c e n t and homogeneous i n t e x t u r e .  The  c o l o u r from dark brown t o gray-green.  d i g e s t i v e gland v a r i e s i n T a b l e 1 i n d i c a t e s t h e range  FIGURE 4 Mean monthly water temperature two f e e t below s u r f a c e at B r o c k t o n Point. V e r t i c a l l i n e s i n d i c a t e t h e range.  FIGURE 5  The p o i n t s r e p r e s e n t t h e average d a y - l i g h t hours p e r day f o r each month, c a l c u l a t e d from t a b l e s - i n N o r t h c o t t (1968, 1969).  21 o f c o l o u r i n t h e d i g e s t i v e gland and t h e number o f animals o f a p a r t i c u l a r c o l o u r i n each c o l l e c t i o n . predominated  i n t h e F e b r u a r y and May  A gray-green d i g e s t i v e gland c o l l e c t i o n s , w h i l e i n August  most animals had yellow-brown d i g e s t i v e g l a n d s . dominantly brown t o dark brown i n t h e A p r i l H i s t o l o g i c a l observations:  The  gland was  pre-  collection.  H i s t o l o g i c a l s e c t i o n s showed t h e  t e s t i s and ovary t o c o n s i s t o f many t u b u l e s i n which t h e gametes appeared  i n v a r i o u s stages o f m a t u r a t i o n .  During r e p r o d u c t i v e p e r i o d s ,  from January t o March, t h e t u b u l e s were l a r g e and c l o s e l y apposed t o each o t h e r but i n n o n - r e p r o d u c t i v e p e r i o d s t h e t u b u l e s were reduced i n s i z e and t h e c o n n e c t i v e t i s s u e spaces between t u b u l e s i n c r e a s e d in size.  The h i s t o l o g i c a l a n a l y s i s o f t h e r e p r o d u c t i v e c y c l e  was  c o n f i n e d t o females o f T. l a m e l l o s a . An attempt  was  made t o c l a s s i f y t h e ovary on a s u b j e c t i v e  scale  r a n g i n g from r i p e t o spent; however, c l e a r c u t changes d i d not  occur  i n t h e ovary o f T. l a m e l l o s a .  Large y o l k g r a n u l e s were p r e s e n t a t  a l l times o f t h e y e a r , as were s m a l l immature o o c y t e s . r e p r o d u c t i v e season, oocytes t h a t remained  A f t e r the  i n t h e ovary appeared  d i s i n t e g r a t e and t h e y o l k g r a n u l e s became s c a t t e r e d throughout tubule.  D e v e l o p i n g oocytes had prominent  n u c l e o l i i n a large  to  the germinal  v e s c i c l e and t h e cytoplasm c o n t a i n e d s m a l l g r a n u l e s .  In l a r g e mature  oocytes t h e n u c l e u s became dense and p y k n o t i c and was  often d i f f i c u l t  t o l o c a t e among t h e l a r g e y o l k g r a n u l e s which c o m p l e t e l y f i l l e d  the  oocyte. D e t e r m i n a t i o n o f r e p r o d u c t i v e c y c l e by oocyte diameter: i n d i c a t e s t h e s i z e d i s t r i b u t i o n o f oocytes i n each c o l l e c t i o n out t h e year.  Each polygon r e p r e s e n t s 125  Figure 6 through-  o o c y t e s , 25 from each  of  22  TABLE 1  Subjective  c l a s s i f i c a t i o n o f d i g e s t i v e gland c o l o u r .  Sample Size  GrayGreen  October 25, 1968  10  -  December 4  11  January 17, 1969  Brown  Dark Brown  8  1  1  1  9  1  -  10  4  4  2  -  F e b r u a r y 16  19  12  4  3  -  April 4  20  5  1  9  5  May 15  20  11  4  4  1  July 1  20  9  7  4  -  August 25  20  —  17  3  _  Collection  Date  YellowBrown  FIGURE 6  Oocyte diameter f r e q u e n c y polygons f o r each monthly c o l l e c t i o n o f T. l a m e l l o s a . Each polygon r e p r e s e n t s a t o t a l o f 125 o o c y t e s and t h e p o i n t s i n d i c a t e t h e per cent o f t h e t o t a l i n each s i z e c l a s s . The a b s c i s s a i s i n per cent u n i t s .  800-900  r  700-800 600-700  sept.  to  25 I 500-600 1968  400-500b\ 300-400 200-300 100-200  o  \  0-100 0  25  PER CENT  FREQUENCY  f i v e females, and each p o i n t i s t h e percentage size class.  of the t o t a l ,  i n that  F o r example, on September 25, 1968, 50% o f t h e oocytes  measured were i n t h e 0 t o 100 u s i z e c l a s s , 22% were i n t h e 100 t o 200  u size class.  500  u and g r e a t e r , o c c u r i n s i g n i f i c a n t ' n u m b e r s from September 1968  to  The graphs i n d i c a t e t h a t oocytes i n t h e range o f  February 1969 and a g a i n i n August 1969.  In A p r i l the s i z e  b u t i o n , shows a l a c k o f t h e l a r g e r s i z e c l a s s e s and p r o b a b l y t h a t spawning has t a k e n p l a c e .  indicates  I n May t h e l a r g e r oocytes b e g i n t o  appear b u t t h e s m a l l s i z e c l a s s e s predominate. appear t o be m a t u r i n g  distri-  I n J u l y t h e oocytes  and i n c r e a s i n g i n s i z e as t h o s e i n t h e s i z e  c l a s s e s g r e a t e r than 300 u b e g i n t o appear i n g r e a t e r numbers.  The  data i n d i c a t e t h a t i n l a t e March o r e a r l y A p r i l most o f t h e p o p u l a t i o n had  spawned and, r e p l e n i s h m e n t  o f l a r g e oocytes began  immediately.  From August t o F e b r u a r y mature o o c y t e s , a t l e a s t from t h e p o i n t o f view o f s i z e , were p r e s e n t i n t h e ovary. Parasites:  I n t h e A p r i l 4 c o l l e c t i o n , 6 out o f 26 T. l a m e l l o s a  examined were i n f e c t e d by t h e p a r a s i t i c trematode o f t h e genus R e n i c o l a ( C h i n g , p e r s . comm.). for  I n f e c t e d T. l a m e l l o s a were n o t used  gonad i n d i c e s o r b i o c h e m i c a l a n a l y s e s . Histology o f d i g e s t i v e gland:  The d i g e s t i v e gland was examined  m i c r o s c o p i c a l l y and t h e number o f s t o r a g e g r a n u l e s i n t h e c e l l s o f t h e d i g e s t i v e gland t u b u l e s was c l a s s i f i e d h a l f f u l l o r empty ( T a b l e 2, P l a t e 1 ) . of  subjectively, f u l l  o f granules,  I t i s assumed t h a t t h e presence  s t o r a g e g r a n u l e s i n d i c a t e s t h a t t h e animals have been f e e d i n g .  If  f e e d i n g s t o p s , t h e g r a n u l e s w i l l g r a d u a l l y d i m i n i s h i n numbers as t h e s t o r e d n u t r i e n t s a r e m o b i l i z e d f o r body maintenance.  The number o f  s t o r a g e g r a n u l e s was a t a maximum i n August and remained f a i r l y h i g h  25  TABLE 2  S u b j e c t i v e c l a s s i f i c a t i o n o f number o f g r a n u l e s i n t h e s t o r a g e o f t h e d i g e s t i v e gland o f T h a i s l a m e l l o s a .  C o l l e c t i o n Date  Sample S i z e  Full  Half-Full  Empty  August 6, 1968  11  10  1  -  September 25  13  11  1  1  October  25  7  7  -  December 4  9  6  3  January 17, 1969  9  6  3  -  February 16  10  -  5  5  April  9  8  1  -  May 15  9  6  1  2  July 1  10  5  3  2  August 25  10  10  Aug. 31 t o J a n . 6 (fed)  8  5  3  -  Aug. 31 t o J a n . 6 (starved)  10  3  5  2  4  Experimental  cells  —  Animals  PLATE 1  C r o s s - s e c t i o n o f d i g e s t i v e gland i l l u s t r a t i n g the s u b j e c t i v e c l a s s i f i c a t i o n o f granule content. 1 = lumen, t = t u b u l e , g = s t o r a g e g r a n u l e . A.  tubules f u l l  o f g r a n u l e s (2600 X)"  B.  tubules h a l f f u l l  C.  t u b u l e s empty (3400 X)  (3400 X)  26 u n t i l October i n the c e l l s ing  25.  From December u n t i l February t h e number o f g r a n u l e s  dropped, p r o b a b l y i n d i c a t i n g t h a t t h e animals were f e e d -  l e s s and u t i l i z i n g s t o r e d n u t r i e n t s .  increased i n A p r i l  and  i n May  The number o f g r a n u l e s  and J u l y t h e numbers dropped a g a i n .  Per Cent Dry Weight The p e r c e n t dry weights  o f t h r e e body organs;  gonad, d i g e s t i v e  g l a n d , and muscle, over a 12 month p e r i o d are p l o t t e d  i n F i g u r e 7.  The per c e n t dry weight o f muscle remained e s s e n t i a l l y c o n s t a n t a t about 27.5%. of  The p e r c e n t dry weight o f ovary ranged  63% i n December t o a minimum o f 40% i n J u l y .  weight o f d i g e s t i v e gland ranged to  from a maximum  The p e r c e n t dry  from a maximum o f 55% i n December  a minimum o f 39% i n J u l y w i t h a f u r t h e r low v a l u e o f 40% i n  February.  The p e r cent dry weight i n d i c a t e s t h e amount o f  solid  m a t e r i a l i n p r o p o r t i o n t o water c o n t e n t ; c o n s e q u e n t l y , when t h e gonads are f u l l o f mature eggs and t h e i n t e r - t u b u l e spaces p e r cent dry weight i s a t a maximum. been f e e d i n g , a l l t h e c e l l s  are s m a l l e s t , t h e  S i m i l a r l y , when t h e animal  has  of t h e d i g e s t i v e gland are f u l l o f s t o r e d  m a t e r i a l and no empty spaces  are e v i d e n t i n t h e h i s t o l o g i c a l s e c t i o n s ,  r e s u l t i n g i n a h i g h p e r c e n t dry  weight.  Gonad Index Another method o f r e p r e s e n t i n g t h e r e p r o d u c t i v e c y c l e i s by the gonad index. females and  The  gonad index c y c l e i s shown i n F i g u r e 8.  Only  the  were sampled i n q u a n t i t i e s which allowed c a l c u l a t i o n o f mean  standard e r r o r .  F o r t h i s p o p u l a t i o n , the ovary index dropped  about 7 d u r i n g t h e months o f October t o F e b r u a r y , p r o b a b l y i n d i c a t i n g spawning. egg s i z e f r e q u e n c y  from  down t o 2 i n A p r i l ,  T h i s c o r r e l a t e s w e l l w i t h t h e data  from  ( F i g . 6) and per cent d r y weight ( F i g . 7) which both  FIGURE 7  Per c e n t o f dry m a t e r i a l i n a u n i t weight o f f r e s h t i s s u e o f T. l a m e l l o s a . The v e r t i c a l l i n e s i n d i c a t e + 1 S.E. Data f o r females o n l y .  1968  MONTH  OF  YEAR  1969  FIGURE 8  Gonad i n d i c e s o f male and female T. l a m e l l o s a as a f u n c t i o n o f month o f y e a r . Closed c i r c l e s ( • ) r e p r e s e n t t h e mean ovary index and open c i r c l e s ( O ) r e p r e s e n t i n d i v i d u a l males V e r t i c a l l i n e s i n d i c a t e + 1 S.E.  20 I o testis  o  • ovary  15 • o o uS  I  o  10 •  0'  °  '  '  A S 1968  '  O  o  o  *  N  '  *  '  '  D J F M A MONTH OF YEAR  '  M  '  J  *  '  J A 1969  1  dropped d u r i n g t h i s same p e r i o d . r o s e a g a i n from 2 up t o 5.5, of  From A p r i l t o May  and t h i s  oocyte s i z e c l a s s e s above 500  is reflected  u i n t h e May  t h e ovary  index  i n t h e appearance  15 c o l l e c t i o n ( F i g . 6).  The t e s t i s index appears t o f o l l o w a s i m i l a r p a t t e r n t o t h e o v a r i e s but w i t h h i g h e r a b s o l u t e v a l u e s d u r i n g t h e w i n t e r months. D i g e s t i v e Gland  Index  The changes i n t h e d i g e s t i v e gland i n d i c e s are shown i n F i g u r e 9. The maximum index o f 22.5  f o r t h e females  and t h e minimum o f 7 i n February.  o c c u r r e d i n l a t e August  1969  The changes i n the d i g e s t i v e gland  index were p a r a l l e l l e d by t h e p e r c e n t d r y weight data i n F i g u r e 7 and t h e s t o r a g e g r a n u l e data i n T a b l e 2.  The maximum index i n August  corresponded  o f s t o r a g e g r a n u l e s , and  t o t h e g r e a t e s t accumulation  minimum index i n F e b r u a r y corresponded content.  t o t h e lowest s t o r a g e  the  granule  The male d i g e s t i v e gland index showed s i m i l a r changes t o t h e  female but due t o t h e s m a l l sample s i z e minor d i f f e r e n c e s c o u l d not be determined. sampling  I t should a l s o be emphasized t h a t due t o t h e method o f  as d e s c r i b e d p r e v i o u s l y , t h e i n d i c e s a r r i v e d a t are o n l y  r e l a t i v e v a l u e s and t h e y underestimate  the t r u e index.  Constituent Levels Digestive gland:  F i g u r e 10 shows t h e l e v e l s o f p r o t e i n ,  lipid,  and glycogen f o r t h e d i g e s t i v e g l a n d ; F i g u r e 11 shows t h e l e v e l s i n t h e f o o t muscle; and F i g u r e 12, t h e gonad. w i l l be c o n s i d e r e d f i r s t . occurred 38.5%  i n t h e female  The  The  changes i n t h e  greatest variation i n l i p i d  The  s t a n t at 6 t o 8%.  lipid  level  d i g e s t i v e gland ( F i g . 10), which went from  i n December t o 20% i n A p r i l , f o l l o w e d by another  35% i n J u l y .  lipid  increase to  l e v e l i n t h e muscle remained e s s e n t i a l l y  The l i p i d  levels  i n t h e male d i g e s t i v e gland  conand  FIGURE 9  D i g e s t i v e gland i n d i c e s o f male and female T. l a m e l l o s a as a f u n c t i o n o f month o f year. Closed c i r c l e s ( • ) represent t h e mean o f 8 females and t h e open c i r c l e s ( o ) r e p r e s e n t i n d i v i d u a l males. V e r t i c a l l i n e s i n d i c a t e + 1 S.E.  FIGURE 10  L e v e l s o f p r o t e i n , l i p i d and glycogen i n t h e d i g e s t i v e gland o f T. l a m e l l o s a as a f u n c t i o n o f month o f t h e y e a r . Each p o i n t r e p r e s e n t s t h e p e r cent o f a c o n s t i t u e n t i n a u n i t weight o f d r y t i s s u e . C l o s e d symbols r e p r e s e n t female means and open symbols r e p r e s e n t i n d i v i d u a l males. V e r t i c a l l i n e s i n d i c a t e + 1 S.E.  Digestive  1968  MONTH  gland  OF  YEAR  1969  32  muscle appeared t o f o l l o w t h o s e o f t h e females.  The ovary l i p i d  f l u c t u a t e d s e v e r a l times d u r i n g t h e y e a r w i t h t h e most  level  significant  change o c c u r r i n g between October and December from 26% down t o 18% (p  <  .05).  f i c a n t (p <  F l u c t u a t i o n s i n F e b r u a r y , A p r i l and May .05).  The t e s t i s l i p i d  were a l s o  signi-  appeared t o drop down t o about  10% i n t h e w i n t e r w i t h p o s s i b l y a s l i g h t r i s e i n J u l y . Protein l e v e l s : o c c u r r e d i n muscle of  46.6%  I n female t i s s u e t h e h i g h e s t p r o t e i n  levels  ( F i g . 11) w i t h a h i g h o f 64% i n A p r i l and a low  i n J u l y 1968.  Ovary was  s l i g h t l y lower w i t h a maximum o f  56% i n October and a g e n e r a l downward t r e n d t o A p r i l where t h e p r o t e i n l e v e l was  46% ( F i g .  12).  The d i g e s t i v e gland showed t h e g r e a t e s t  range i n p r o t e i n l e v e l from a maximum o f 43.5% i n A p r i l t o a minimum of  31% i n F e b r u a r y .  The p r o t e i n l e v e l s i n male muscle and  digestive  glands were, as near as c o u l d be determined by t h e s m a l l sample the  same; but i n t h e t e s t i s , p r o t e i n v a l u e s were g e n e r a l l y lower and  averaged about  40%.  Glycogen and ash l e v e l s :  Emerson (1966) showed t h a t t h e p o l y -  s a c c h a r i d e o f T. l a m e l l o s a i s 100% g l y c o g e n ; hence, p o l y s a c c h a r i d e w i l l be r e f e r r e d t o as glycogen. low l e v e l s 4%;  size,  i n a l l three tissues.  i n t h e muscle  Glycogen was  at very  In t h e ovary i t ranged from 2% t o  i t f l u c t u a t e d from 2% t o 4%;  gland t h e range was  i n t h i s study t h e  and i n t h e d i g e s t i v e  s l i g h t l y g r e a t e r , from 2% t o 7.5%.  g l y c o g e n l e v e l i n t h e male t i s s u e s was  Again, the  e s s e n t i a l l y t h e same.  Ash  weights f o r t h e t h r e e t i s s u e s a t t h r e e times d u r i n g t h e y e a r are r e p r e s e n t e d i n F i g u r e 13.  Male and female t i s s u e s were combined  in a l l  FIGURE 11  L e v e l s o f p r o t e i n , l i p i d and glycogen i n t h e f o o t muscle o f T. l a m e l l o s a as a f u n c t i o n o f month o f y e a r . Each p o i n t r e p r e s e n t s t h e p e r cent o f a c o n s t i t u e n t i n a u n i t weight o f d r y t i s s u e . C l o s e d symbols r e p r e s e n t female means and open symbols r e p r e s e n t i n d i v i d u a l males. Vert i c a l l i n e s i n d i c a t e + 1 S.E.  80 Foot muscle 70 ox  60  50h  to  6  40  lipid  10 h  A  o o  .2 glycogen  1 A  S  1968  O  N  D  J  F  M  MONTH OF YEAR  i  i  M  J 1969  J  FIGURE 12  L e v e l s o f p r o t e i n , l i p i d and glycogen i n t h e gonad o f T. l a m e l l o s a as a f u n c t i o n o f month o f y e a r . Each p o i n t r e p r e s e n t s t h e p e r cent o f a c o n s t i t u e n t i n a u n i t weight o f d r y t i s s u e . C l o s e d symbols r e p r e s e n t female means and open symbols r e p r e s e n t i n d i v i d u a l males. Vertical l i n e s i n d i c a t e + 1 S.E.  A  S  1968  O  N  D  J  F  M  MONTH OF  YEAR  A  M  J  1969  J  A  35 cases except t h e gonad which r e p r e s e n t s o n l y the females. muscle and  gonad have s i m i l a r ash l e v e l s a t t h e t h r e e  Both  collection  d a t e s ; however, t h e ash l e v e l i n t h e d i g e s t i v e gland r i s e s from  3.4%  i n August t o 7% i n March. F i g u r e 14 shows t h e amount o f dry weight accounted  f o r by  lipid,  glycogen, and p r o t e i n a n a l y s e s i n t h e d i g e s t i v e gland each month. In  September, January  It  can be seen t h a t t h e s e a n a l y s e s account f o r o n l y about 80% o f the  dry  and May,  the ash weight v a l u e s have been added.  weight.  Experimental Results As d e s c r i b e d i n t h e methods s e c t i o n , two were m a i n t a i n e d January  6, 1969}  groups o f T. l a m e l l o s a  i n summer c o n d i t i o n s from August 31, 1968 one  group b e i n g f e d , t h e o t h e r , s t a r v e d .  c o l l e c t i o n , made a t t h e b e g i n n i n g o f t h e experiment l o c a t i o n (Aug. lipid,  until A regular  from t h e same  31) served t o e s t a b l i s h the s t a r t i n g l e v e l s o f p r o t e i n ,  glycogen and  dry weight.  The  e x p e r i m e n t a l animals  at t h e  were assumed t o have t h e same b i o c h e m i c a l l e v e l s as t h e f i e l d  start  group  s i n c e t h e y were c o l l e c t e d from t h e same p o p u l a t i o n .  A t t h e end  t h e e x p e r i m e n t a l p e r i o d another f i e l d  a n a l y s e d ; hence,  t h e changes i n t h e normal animals determined.  A t t h e end  c o l l e c t i o n was  d u r i n g t h e e x p e r i m e n t a l p e r i o d were  o f t h e experiment,  t h e f e d and t h e s t a r v e d  groups were a n a l y s e d f o r t h e v a r i o u s b i o c h e m i c a l c o n s t i t u e n t s . 3 summarizes t h e d a t a , g i v i n g l e v e l s o f l i p i d , weight and component i n d i c e s f o r t h e f i e l d m e n t a l groups.  The  of  Table  glycogen, p r o t e i n ,  dry  c o n t r o l s and t h e two e x p e r i -  s t a r t i n g v a l u e s f o r t h e dry weight l e v e l and com-  ponent i n d i c e s were not o b t a i n e d on August 31 so t h e data from September 25 c o l l e c t i o n were used  as s t a r t i n g v a l u e s .  the  FIGURE 13 P e r cent ash p e r u n i t weight o f dry t i s s u e i n t h r e e t i s s u e s o f t h e body. The data f o r d i g e s t i v e gland and f o o t muscle a r e means o f pooled male and female t i s s u e .  10  digestive gland foot muscle ovary i i  a  i  |  li  JAN ' 6 9  i I  MAY ' 6 9  FIGURE  14  Histograms r e p r e s e n t t h e amount o f dry t i s s u e accounted f o r by t h e p r o t e i n , l i p i d and glycogen d e t e r m i n a t i o n s . Data f o r ash i s a v a i l a b l e f o r only three of the c o l l e c t i o n s .  Digestive gland HUM i — i  §  s  §  I  B  s S  : B S  I i  §  fi  50 •  • s  8 8 S  5  I  I  A  11 1 N S  1968  glycogen lipid protein ash  I I  D  J  F  •I i MONTH OF• YEAR  M  LA  A  M  i J 1969  J  38  TABLE 3  Summary o f data f o r two e x p e r i m e n t a l groups o f T. l a m e l l o s a m a i n t a i n e d i n summer c o n d i t i o n s from August 31, 1968 t o J a n u a r y 6, 1969. Data f o r females o n l y . Numbers i n b r a c k e t s a r e 1 S.E. c^ = f i e l d c^  =  sample a t s t a r t  field  o f experiment (August 31).  sample a t end o f experiment (January 6 ) .  f  = e x p e r i m e n t a l group f e d , and sampled a t c o n c l u s i o n .  s  = e x p e r i m e n t a l group s t a r v e d , and sampled a t c o n c l u s i o n .  Tissue  Digestive Gland  Ovary  Foot Muscle  Lipid  Glycogen  Protein  %  Dry Weight  Component Index  21.8(+1.2)  6.6(+0.9)  42.4(+0.7)  46.6(+3.0) (Sept. 25)  13.9(+1.0) (Sept. 25)  33.0(+2.9)  3.4(+0.4)  37.7(0.9)  47.9(2.6)  10.8(1.1)  f  32.9(+2.l)  7.0(0.6)  37.7(1.9)  46.2(2.2)  13.8(1.0)  s  35.6(+1.9)  6.3(0.9)  33.8(1.9)  43.5(1.5)  12.4(1.1)  20.7(+1.0)  3.6(0.5)  48.8(1.4)  52.6(4.3)  5.5(0.6)  . 22.5(+1.0)  3.0(0.2)  49.9(0.7)  61.5(0.6)  6.6(0.6)  f  31.4(+1.1)  4.2(0.5)  42.3(1.3)  54.4(3.4)  4.1(0.8)  s  32.5(+1.9)  4.0(0.4)  42.2(2.1)  52.1(2.9)  2.5(0.4)  6.7(+0.2)  3.5(0.3)  55.7(1.3)  27.0(0.2)  8.1(+0.1)  2.3(0.3)  56.2(0.7)  26.9(0.5)  f  6.7(+0.3)  4.2(0.6)  60.4(1.0)  25.6(0.7)  s  6.6(+0.2)  3.4(0.2)  53.6(1.4)  24.0(0.6)  C  l  C  2  C  l  C  2  C  l  C  2  The d i g e s t i v e gland index o f t h e f i e l d 13.9  t o 10.8  (p <  p o p u l a t i o n dropped  from  .05) d u r i n g t h e e x p e r i m e n t a l p e r i o d ( T a b l e 3).  However, t h e d i g e s t i v e gland i n d i c e s o f the f e d and s t a r v e d animals did  not change.  T a b l e 2 shows t h e s t o r a g e g r a n u l e data f o r t h e  e x p e r i m e n t a l groups.  two  Most o f t h e f e d i n d i v i d u a l s were f u l l o f s t o r a g e  g r a n u l e s , w h i l e the d i g e s t i v e glands o f t h e m a j o r i t y o f s t a r v e d animals were h a l f - f u l l o f g r a n u l e s .  There was  c r e a s e i n t h e number o f mucous c e l l s  i n s t a r v e d animals.  The mean ovary index o f t h e f i e l d  also a noticeable i n -  p o p u l a t i o n and t h e f e d group d i d  not change s i g n i f i c a n t l y d u r i n g t h e e x p e r i m e n t a l p e r i o d . t h e r e was  a s i g n i f i c a n t drop i n t h e ovary index o f t h e s t a r v e d group  from 5.5 t o 2.5 of  However,  (p <. .05).  F i g u r e 15 shows t h e p e r cent f r e q u e n c y  oocytes i n each s i z e c l a s s f o r t h e two  e x p e r i m e n t a l groups.  In both  cases more t h a n 75% o f t h e oocytes measured are i n t h e range o f 0 t o 100 microns, and none o c c u r i n t h e c l a s s e s g r e a t e r than 500 In  the d i g e s t i v e glands o f t h e f i e l d  glycogen l e v e l s dropped  (p <~ .05), l i p i d  t h e dry weight l e v e l remained p r o t e i n l e v e l dropped  constant.  s i g n i f i c a n t l y (p <1  u.  c o n t r o l s the p r o t e i n i n c r e a s e d (p  and  .05), and  In t h e f e d i n d i v i d u a l s t h e .05) and l i p i d i n c r e a s e d  s i g n i f i c a n t l y (p -<i .05), w h i l e g l y c o g e n and dry weight remained same.  the  As i n t h e f e d i n d i v i d u a l s t h e d i g e s t i v e glands o f t h e s t a r v e d  animals showed a s i g n i f i c a n t drop i n p r o t e i n (p <C in l i p i d  .05), an i n c r e a s e  (p <: .05) and no change i n glycogen o r dry  The o v a r i e s o f t h e f i e l d the l i p i d ,  weight.  c o n t r o l s showed no s i g n i f i c a n t change i n  p r o t e i n , o r glycogen l e v e l s .  In both t h e f e d and s t a r v e d  animals s i g n i f i c a n t changes o c c u r r e d i n l i p i d  and p r o t e i n (p <L  .05)  FIGURE 15  Oocyte diameter f r e q u e n c y polygons f o r two e x p e r i m e n t a l groups. E x p l a n a t i o n as i n F i g u r e 6. Animals k e p t under summer c o n d i t i o n s from August 31, 1968 u n t i l January 6, 1969.  g 600-700  fed  o  1 500-600 400-500 < 300-400 m  200-300  i —  y  100-200  \  o  0-100 0  25  PER CENT FREQUENCY  but glycogen and d r y weight remained t h e same. D u r i n g t h e e x p e r i m e n t a l p e r i o d t h e muscle t i s s u e o f t h e animals showed s m a l l changes i n t h e l i p i d were s i g n i f i c a n t a t t h e 5% l e v e l . individuals,  lipid  field  and glycogen l e v e l s which  In t h e muscle t i s s u e o f t h e f e d  and glycogen l e v e l s remained t h e same but t h e  p r o t e i n l e v e l i n c r e a s e d s i g n i f i c a n t l y (p <  .05).  The s t a r v e d i n -  d i v i d u a l s on t h e o t h e r hand, showed no change i n p r o t e i n , l i p i d , glycogen  levels.  or  42 DISCUSSION  R e p r o d u c t i o n and Development In  a l l h i g h e r prosobranchs, f e r t i l i z a t i o n  (Thorson, 1950).  The f e r t i l i z e d  ment i n one o f s e v e r a l ways.  i s by c o p u l a t i o n  eggs t h e n proceed through  develop-  They may e i t h e r develop i n s i d e t h e  female by v i v i p a r i t y o r some form o f i n t e r n a l brood p r o t e c t i o n ; t h e y may be shed s i n g l y i n t o t h e water and proceed through development as p e l a g i c l a r v a e ; t h e y may develop i n a g e l a t i n o u s mass o r s t r i n g ; or,  as i n T h a i s l a m e l l o s a , be d e p o s i t e d on t h e s u b s t r a t e w i t h i n a  c a p s u l e (Thorson, 1946).  Larvae which develop i n s i d e c a p s u l e s l e a v e  a f t e r a c e r t a i n p e r i o d and complete  development as p e l a g i c o r non-  p e l a g i c young. The eggs o f T. l a m e l l o s a , when l a i d , a r e i n t h e o r d e r o f 500 microns  i n diameter.  T h i s l a r g e s i z e i s a p p a r e n t l y i n d i c a t i v e o f non-  p e l a g i c development (Thorson, 1946).  Some examples o f egg s i z e s f o r  s p e c i e s w i t h n o n - p e l a g i c development d e s c r i b e d by Thorson, a r e Trophon h a n l e y i (500 u ) , Brachystomia r i s s o i d e s (380 u ) , Amauropsis (1500  islandica  u ) , and Neptunea a n t i q u a (300 u ) . Development w i t h i n a c a p s u l e  has s e v e r a l advantages..  S i n c e every egg d e p o s i t e d i n t h e c a p s u l e i s  f e r t i l i z e d , t h e r e i s l e s s "waste" t h a n i n b r o a d c a s t  fertilization.  The l a r g e s u p p l y o f y o l k enables t h e l a r v a t o d e v e l o p t o an advanced s t a g e w i t h i n t h e c a p s u l e , t h e r e b y r e d u c i n g p r e d a t i o n encountered by pelagic species.  Thorson  r e l a t i v e advantages  (1950) d i s c u s s e s t o a g r e a t e r e x t e n t t h e  o f t h e d i f f e r e n t t y p e s o f l a r v a l development i n  r e l a t i o n t o t h e environmental c o n d i t i o n s . The c a p s u l e s o f some s p e c i e s c o n t a i n n u r s e eggs, which a r e  oocytes t h a t do not develop but become a food source f o r t h e o t h e r l a r v a e ( F r e t t e r , 1941).  The  stage o f development a t h a t c h i n g  reached  by t h e l a r v a e which u t i l i z e nurse eggs, v a r i e s g r e a t l y , depending on t h e r a t i o o f nurse eggs t o l a r v a e .  Embryos w i l l l e a v e t h e c a p s u l e  when t h e y have reached t h e stage o f development t h a t t h e nurse w i l l support. may  Because o f t h i s , embryos o f a s p e c i e s i n one  eggs  locality  h a t c h as n o n - p e l a g i c young w h i l e i n another l o c a l i t y where fewer  nurse eggs were produced, t h e embryos may (Thorson, 1950).  h a t c h as p e l a g i c l a r v a e  T. l a m e l l o s a does not produce nurse eggs (Ahmed and  Sparks, 1970); hence t h e l a r v a e always l e a v e t h e c a p s u l e a t t h e same stage o f development.  Emlen (1966) found t h a t egg-capsules  T. l a m e l l o s a c o n t a i n e d from 20 t o 150 e s t i m a t e d t h a t a female  eggs w i t h a mean o f 81.  can l a y about 200 c a p s u l e s a y e a r and  i n g a spawning p e r i o d o f 5 months, 1.35 The  of  capsules  He  also  assum-  per-day.  eggs o f T. l a m e l l o s a are l a r g e r and more y o l k y t h a n many  o t h e r s p e c i e s , so oogenesis  i s probably a longer process.  It is  r e a s o n a b l e t o expect t h e n , t h a t T. l a m e l l o s a spawns o n l y once a y e a r , w h i l e some b r o a d c a s t f e r t i l i z e r s which produce much s m a l l e r gametes, may  spawn more o f t e n .  The b i v a l v e , S p i s u l a s o l i d i s s i m a (Ropes,  and t h e h a r d - s h e l l clam, Venus m e r c e n a r i a have two  spawning p e r i o d s i n a y e a r .  (1969), t h e abalone, f e r t i l i z e r , had two t h e r e was  Lander,  1967)  A c c o r d i n g t o Webber and  Giese  H a l i o t i s c r a c h e r o d i i , which i s a b r o a d c a s t p e r i o d s o f gametogenesis i n t h e y e a r  p r o b a b l y o n l y one  spawning p e r i o d .  i n c r e a s e i n s i z e o n l y d u r i n g one gametogenic p e r i o d , which was may  ( A n s e l l and  1968)  although  The ovary showed an  gametogenic p e r i o d .  The  second  s a i d t o have o c c u r r e d a f t e r spawning,  have been an a r t i f a c t o f t h e sampling t e c h n i q u e , as e x p l a i n e d  below.  In t h e present  summer ( F i g . 6 ) , and The  study,  one  gametogenesis o c c u r r e d  spawning peak o c c u r r e d  l a t e r In t h e  i n March and  April.  sudden i n c r e a s e i n t h e s m a l l e r s i z e c l a s s e s i n A p r i l may  have n e c e s s a r i l y been due  t o an  immediate i n c r e a s e i n t h e i r numbers  but r a t h e r t o a l o s s o f t h e l a r g e mature o o c y t e s . percentages;  hence i f no l a r g e oocytes  s m a l l oocytes  appears t o i n c r e a s e .  c l u s i o n by Webber and period  Giese  not  The  data are i n  are p r e s e n t t h e percentage o f  T h i s perhaps e x p l a i n s t h e  con-  (1969) t h a t t h e r e i s a second gametogenic  immediately a f t e r spawning i n H. c r a c h e r o d i i .  Emlen (1966) determined t h e developmental time w i t h i n t h e o f T. l a m e l l o s a t o be 140  days.  P o i n t p o p u l a t i o n t h e eggs l a i d  I f t h a t i s t r u e f o r the i n March would h a t c h  s u b j e c t i v e e s t i m a t e , made on J u l y 1, 1969 40 p e r cent o f t h e c a p s u l e s  Brockton  i n mid-summer.  checked were empty.  1965.  Animals were s t i l l  by A p r i l 21, present  on November 20  1966.  The  i n 1964  Emlen (1966),  and  spawning on March 12,  breeding  1966  s a l i n i t y and  have been s l i g h t l y l a t e r . more c o n s t a n t  temperature, s i n c e t h e area  extent  i n the the The  begincon-  i s more d i r e c t l y  Influenced  The  exact  o f spawning by T. l a m e l l o s a at Brockton P o i n t  not determined by e x t e n s i v e f i e l d  observations,  v a t i o n a t t h e time o f r e g u l a r sampling. were l a i d  ceased  with respect t o  by t h e P a c i f i c Ocean through t h e Juan de Fuca S t r a i t . p e r i o d and  first  and had  period f o r the population  d i t i o n s a t P o r t Townsend are p r o b a b l y  study-  on December 10 i n  study agrees with t h a t r e p o r t e d by Emlen, a l t h o u g h  n i n g o f t h e c a p s u l e l a y i n g may  A  i n d i c a t e d t h a t about 30 t o  i n g a p o p u l a t i o n at P o r t Townsend, Washington, r e p o r t e d t h e appearance o f c a p s u l e s  capsules  but merely by  I t i s possible that  at v e r y low t i d a l l e v e l s ( z e r o f e e t ) b e f o r e those  was  obsercapsules  first  45 observed i n J a n u a r y , 1969. P o r t Townsend was 45 and 50 mm One  60 mm  The average s i z e o f T. l a m e l l o s a from  w h i l e t h o s e from Brockton P o i n t averaged  between  i n length.  i n d i v i d u a l o f T. l a m e l l o s a c o l l e c t e d on October 25,  1968,  and kept i n an aquarium, l a i d t h r e e egg-capsules on November 16, Whether t h i s difficult  i n c i d e n t can be extended t o t h e f i e l d  t o say.  A few i n d i v i d u a l s i n t h e f i e l d  u l a t i n g on December 4, 1968 p l a c e soon It  1968.  population i s were observed  so c o n c e i v a b l y , e g g - l a y i n g may  cop-  have taken  after.  i s assumed t h a t an oocyte r e q u i r e s one y e a r t o mature; however,  t h e r e i s no d i r e c t evidence t o support t h i s c o n c l u s i o n .  N i m i t z and  G i e s e (1964) r e p o r t t h a t oogenesis i n K a t h e r i n a t u n i c a t a i s a two p r o c e s s , but t h e y do n o t o f f e r evidence t o support t h i s  year  statement.  The egg diameter f r e q u e n c y data ( F i g . 6) i n d i c a t e t h a t mature s i z e oocytes (500 a) are p r e s e n t i n t h e ovary from September t o t h e time of t h e F e b r u a r y c o l l e c t i o n .  Furthermore,  t h e gonad i n d i c e s  d u r i n g t h i s t i m e are e s s e n t i a l l y t h e same.  ( F i g . 8)  On t h e b a s i s o f oocyte  size  and gonad index, i t appears t h a t T. l a m e l l o s a has t h e p o t e n t i a l t o spawn d u r i n g t h e p e r i o d from September t o A p r i l . There are p o s s i b l y two  p e r i o d s o f s t i m u l a t i o n which may  the t i m i n g of the breeding c y c l e .  The f i r s t induces  affect  gametogenesis  and oocyte development, and t h e second t r i g g e r s a c t u a l spawning o r egg l a y i n g .  Thorson  (1946) m a i n t a i n s t h a t t h e inducement f o r gameto-  g e n e s i s i s a temperature ing;  d i f f e r e n t from t h a t r e q u i r e d t o induce spawn-  hence a t t h e edge o f a s p e c i e s ' g e o g r a p h i c a l range an  animal  c o u l d c o n c e i v a b l y r i p e n i t s s e x u a l p r o d u c t s but n o t spawn them.  Orton,  Southward, and Dodd (1956) s t u d y i n g t h e l i m p e t , P a t e l l a v u l g a t a , con-  46 eluded t h a t t h e breeding c y c l e o f a population i s r e l a t i v e l y  constant  from y e a r t o y e a r ; t h a t i s , t h e p o p u l a t i o n i s p o t e n t i a l l y ready t o spawn d u r i n g a s p e c i f i c time i n t h e y e a r , e.g. September t o A p r i l , but t h e spawning s t i m u l u s seems t o o v e r - r i d e t h e b r e e d i n g  cycle.  Depending on t h e y e a r o r t h e l o c a l i t y , t h e time o f t h e r e l e a s e o f gametes may be q u i t e v a r i a b l e . appeared or  I n t h e case o f P. v u l g a t a spawning  t o correspond t o rough seas r a t h e r than t o temperature,  tides,  phases o f t h e moon (Orton^ e t a l . , 1956).  S e a s o n a l B i o c h e m i c a l Changes The r e s u l t s o f t h e e x p e r i m e n t a t i o n i n t h i s study suggest t h a t temperature,  l i g h t , o r s a l i n i t y r a t h e r t h a n food might induce gameto-  g e n e s i s and egg l a y i n g i n T. l a m e l l o s a , b u t i t w i l l be n e c e s s a r y t o perform c o n t r o l l e d experiments are i n v o l v e d .  i n o r d e r t o conclude which  stimuli  The work p r e s e n t e d h e r e d e s c r i b e s t h e b i o c h e m i c a l  changes which t o o k p l a c e i n t h i s animal d u r i n g t h e b r e e d i n g c y c l e o f 1968-69. of  From t h e s e d a t a , c o n c l u s i o n s w i l l be drawn about t h e t r a n s f e r  s t o r a g e p r o d u c t s w i t h i n t h e v a r i o u s organs o f t h e s n a i l , and t h e  r e l a t i o n s h i p s among f e e d i n g , gametogenesis, spawning and t h e b i o chemical l e v e l s .  B i o c h e m i c a l a n a l y s e s were performed  on whole t i s s u e s ;  hence any i n t e r n a l t r a n s f e r o f c o n s t i t u e n t s c o u l d n o t be measured b u t o n l y suggested.  H i s t o c h e m i c a l data from r e l a t e d s p e c i e s o f m o l l u s k s  and t h e m o r p h o l o g i c a l changes observed to  i n t h i s study w i l l be employed  e x p l a i n t h e gross b i o c h e m i c a l changes. Biochemical constituents o f oocytes:  t y p e s o f y o l k formed d u r i n g v i t e l l o g e n e s i s  Raven (1966) reviewed t h e i n mollusks.  In the nudi-  branch, A p l y s i a and t h e pulmonate s n a i l s , Limnaea, H e l i x , and Eremina, t h e f i r s t t o be formed was p r o t e i n y o l k and i n l a t e r stages l i p i d was  47 added.  Nudibranch eggs are 66% t o 75% by volume p r o t e i n y o l k  o n l y about 2% t o 5% l i p i d may be  have l i p i d  yolk.  However, some o f the p r o t e i n  associated with i t .  Crepidula  75% p r o t e i n y o l k (Raven, 1966).  L i p i d provides  C r e p i d u l a , but  Smith, 1964).  c o n s t i t u t e n t s i n T. l a m e l l o s a  longer  The  eggs were not  o f the eggs o f N u c e l l a l a p i l l u s ,  levels deter-  i n the  a closely related  c o n s t i t u e n t s t h e animal may  oocytes with t h e r e q u i r e d i n g e s t food  t h e gonad as i n S t r o n g y l o c e n t r o t u s s t o r e food to the  reserves  i n the p y l o r i c caeca and  t r a n s f e r s them t o t h e  (Farmanfarmaian et a l . , 1958).  B. balanus s t o r e m a t e r i a l d u r i n g the  and  t r a n s f e r i t t o the  gonads  Balanus  s p r i n g when food  during  i s abundant  gonads i n the w i n t e r (Barnes et a l . , 1963).  of ingested  (Yonge, 1937;  food  balanoides  I t i s g e n e r a l l y accepted t h a t the c h i e f s i t e o f  storage  to  et a l . , 1959a),  P i s a s t e r ochraceus s t o r e s  and  Morton, 1951).  transfer i t directly  purpuratus (Giese  gonad when r e q u i r e d .  the breeding period  and  biochemical  i n another p a r t o f the body and t r a n s f e r the  reserves  Feeding:  and  essential constituents.  In o r d e r t o supply t h e  gland  eggs  F o r the m a t u r a t i o n o f o o c y t e s , i t appears t h a t p r o t e i n  are the  t i o n and  s i n c e the  Bayne (1968) found t h a t f r e e l i p i d s were p r e s e n t  n u t r i e n t reserve  o r i t may  probably  more than t w i c e as many c a l o r i e s per gram t h a n e i t h e r  of the biochemical  lipid  to  s p e c i e s , i t would seem economical t o add more l i p i d .  c a r b o h y d r a t e o r p r o t e i n (White, Handler, and  species.  eggs are  t h e l a r v a e must l i v e o f f the y o l k c o n t e n t f o r a  t i m e than most o t h e r  mined, but  yolk  eggs are r e p o r t e d  T. l a m e l l o s a  s i m i l a r t o t h o s e o f nudibranchs and are l a r g e r and  and  food  M i l l o t t , 1938;  i n gastropods i s the Graham, 1939;  digestive  Howells, 1942;  In t h i s study, the presence o f s t o r a g e  diges-  and  granules i n  48 t h e d i g e s t i v e gland T h i s method was  d i v e r t i c u l a was  used by Mauzey (1966) as an i n d i c a t i o n  P i s a s t e r ochraceus.  Table  i n August, September, and December and January. age  granules  used as an i n d i c a t i o n  was  of feeding.  of feeding i n  2 i n d i c a t e s t h a t T. l a m e l l o s a was October, 1968  and t o a l e s s e r  feeding  extent  in  In t h e F e b r u a r y c o l l e c t i o n , t h e number o f  least,  stor-  s u g g e s t i n g t h e l e a s t amount o f f e e d i n g .  This  p e r i o d corresponded t o t h e b r e e d i n g time when most o f t h e s n a i l s were a t t h e zero t i d e l e v e l forming  breeding  sumably not  Thorson (1958) documents " p a s s i v e  a c t i v e l y feeding.  aggregations,  and  were p r e -  periods" i n s e v e r a l predator  species of bottom-living invertebrates,  especially  In t h e A p r i l c o l l e c t i o n , when most i n d i v i -  d u a l s had  during breeding.  completed spawning, t h e i n c r e a s e i n s t o r a g e  t h a t f e e d i n g had  resumed.  The  decrease o f s t o r a g e  and J u l y 1 imply t h a t f e e d i n g was  reduced.  granules  granules  on May  and  As mentioned i n t h e  site  I n t r o d u c t i o n , Chapman  Banner (1949) found t h a t reduced s a l i n i t y (l5°/oo) caused a  r e d u c t i o n i n m o t i l i t y and  even w i t h d r a w a l i n t o t h e s h e l l ;  i n d i r e c t l y reducing feeding.  The  not t e s t e d , although  the suggestion  (Johannsson, p e r s . comm.).  The  conclusions  index  present  present  work on t h i s aspect  about f e e d i n g , based on t h e s t o r a g e  t e n t , are a l s o supported  thereby  e f f e c t o f s a l i n i t y on t h e  p o p u l a t i o n was  The  15  A reduction i n feeding  might be e x p l a i n e d by a decrease i n s a l i n i t y at t h e c o l l e c t i o n due t o r u n - o f f ( F i g . 2).  suggest  by t h e d i g e s t i v e gland  supports  granule  con-  i n d i c e s ( F i g . 9).  drops from October u n t i l February t h e n r i s e s i n A p r i l when  f e e d i n g resumes a f t e r spawning. t h e p e r i o d o f low  s a l i n i t y i n May  i n t h e l a t t e r p a r t o f t h e summer.  There was  a drop i n t h e  index  during  and June, f o l l o w e d by a l a r g e i n c r e a s e The  index  showed an i n c r e a s e c o r -  49 r e s p o n d i n g t o t h e i n c r e a s e i n t h e number o f s t o r a g e E x p l a n a t i o n o f terms;  granules.  Before d i s c u s s i n g t h e data on  changes, s e v e r a l p o i n t s should be made c l e a r .  When p r e s e n t i n g t h e  l e v e l s o f p o l y s a c c h a r i d e i n T. l a m e l l o s a , determined work, t h e y w i l l be r e f e r r e d t o as glycogen,  biochemical  i n the  present  s i n c e Emerson (1965) has  shown by chromatography t h a t t h e p o l y s a c c h a r i d e i n T. l a m e l l o s a i s 100  per c e n t glycogen.  However, when r e f e r e n c e i s made t o o t h e r  papers, t h e g e n e r a l term p o l y s a c c h a r i d e w i l l be used u n l e s s author s t a t e d t h e s p e c i f i c p o l y s a c c h a r i d e which was The  constituent levels  the  measured.  i n t h i s study are expressed  as p e r c e n t  o f a u n i t weight o f dry t i s s u e ; t h e r e f o r e care should be e x e r c i s e d i n i n t e r p r e t i n g the d a t a .  F o r example, i f one  from a t i s s u e , t h e r e m a i n i n g appear t o i n c r e a s e .  The  constituent i s u t i l i z e d  c o n s t i t u e n t s , on a p e r cent b a s i s ,  change i n s i z e o f an organ must be  when i n t e r p r e t i n g t h e p e r cent d a t a . i n c o n j u n c t i o n w i t h a decrease  may  considered  Changes i n c o n s t i t u e n t l e v e l s  i n organ s i z e , may  i n d i c a t e t h a t the  c o n s t i t u e n t s have a l l been u t i l i z e d but a t d i f f e r e n t r a t e s . data t h e n , when c o n s i d e r e d w i t h changes i n organ s i z e , can  Per cent indicate  approximate r a t e s o f u t i l i z a t i o n . Constituents of d i g e s t i v e glandi  I f i n g e s t e d food  is first  stored  i n the d i g e s t i v e g l a n d , t h e r e should be a p a r a l l e l i n c r e a s e i n one more o f t h e b i o c h e m i c a l c o n s t i t u e n t s and the d i g e s t i v e gland  or  index.  I f t h e c o n s t i t u e n t l e v e l s i n the gland ( F i g . 10) a r e compared t o t h e d i g e s t i v e gland and  glycogen  i n d i c e s ( F i g . 9 ) , i t w i l l be noted t h a t t h e p r o t e i n  l e v e l s peak at times c o r r e s p o n d i n g t o t h e index peaks,  e s p e c i a l l y i n A p r i l and August, 1969. peak i n September, 1969,  There was  but no d i g e s t i v e gland  a p r o t e i n and index was  glycogen  obtained.  50 L i p i d l e v e l s peak about 3 months a f t e r t h e September and A p r i l peaks, on December 4, 1968 and J u l y 1, 1969, r e s p e c t i v e l y . August 31, 1969, t h e l i p i d l e v e l began t o decrease.  S t a r t i n g on  l e v e l began t o i n c r e a s e and t h e glycogen S i n c e f e e d i n g was s t i l l  o c c u r r i n g through  August, September, and October, and t h e d i g e s t i v e gland was e n l a r g i n g , t h e food was most l i k e l y b e i n g s t o r e d as l i p i d . in p r o t e i n i s probably  The apparent drop  a passive r e s u l t o f the increase i n l i p i d .  P r o t e i n makes up t h e s t r u c t u r a l p a r t s o f t i s s u e s ; hence an i n c r e a s e i n s t o r e d m a t e r i a l , such as l i p i d , on a p e r cent b a s i s .  w i l l result  As a r u l e , t h e r e  i n a drop i n p r o t e i n  i s a net l o s s o f p r o t e i n only  under s t a r v a t i o n c o n d i t i o n s (White, Handler, and Smith, 1964). f a c t t h a t t h e p r o t e i n curve lipid  curve  probably  i n F i g u r e 10 i s t h e r e c i p r o c a l o f t h e  i n d i c a t e s the passive nature  changes i n r e l a t i o n t o t h e s t o r e d The  biochemical  The  composition  of the protein  lipid. o f b a r n a c l e s , t h e food o f T. l a m e l l o s a ,  based on t h e data from Barnes e t a l . (1963) f o r two s p e c i e s o f Balanus, show t h a t t h e r a t i o o f l i p i d 2:1.  I f the barnacles  t i o n t o those  composi-  r e p o r t e d by Barnes e t a l . (1963) t h e n t h e s n a i l s  t h e d i g e s t i v e gland  to lipid  as p o l y s a c c h a r i d e .  level i n level  T h i s suggests t h a t i n g e s t e d glycogen i s  f a i r l y quickly, at least  animal i s preparing f o r reproduction.  i n t h e f a l l when t h e  During t h e summer, when food  i s p l e n t i f u l , t h e animal may u t i l i z e glycogen r a t h e r than convert  The glycogen  will  does n o t reach more t h a n 7%, w h i l e t h e l i p i d  does n o t f a l l below 20%.  great, l i p i d  i s about  eaten by T. l a m e l l o s a have a s i m i l a r  i n g e s t t w i c e as much l i p i d  converted  t o polysaccharide  i t to lipid.  i s a t i t s lowest  d i r e c t l y f o r energy  I n l a t e August, when f e e d i n g i s  l e v e l and glycogen  i s a t a maximum, but  51 as f a l l and w i n t e r approach,  r e p r o d u c t i v e demands i n c r e a s e and  lipid  becomes more prominent i n t h e s n a i l ' s economy. Emerson (1965) analysed s e v e r a l P a c i f i c west c o a s t for  t h e i r p o l y s a c c h a r i d e c o n t e n t and found  a positive  prosobranchs  correlation  between t h o s e w i t h a c a r n i v o r o u s d i e t and t h e l a r g e s t p o l y s a c c h a r i d e content.  He r e p o r t e d a glycogen l e v e l o f 6.05  ( + 1 . 1 6 , 95%  confidence  l i m i t ) per c e n t o f t h e dry weight f o r t h e combined s o f t p a r t s o f T. l a m e l l o s a c o l l e c t e d  i n July-August.  T h i s glycogen l e v e l r e p o r t e d by  Emerson i s i n t h e same range as l e v e l s i n t h i s study, but a d i r e c t comp a r i s o n cannot be made, s i n c e h i s d e t e r m i n a t i o n s were performed t o t a l 'soft p a r t s r a t h e r t h a n on i n d i v i d u a l t i s s u e s . prosobranchs  The  herbivorous  had p o l y s a c c h a r i d e v a l u e s i n t h e range o f 1 t o 3 p e r c e n t .  Emerson (1965) a l s o determined  the t o t a l ether extractable m a t e r i a l  i n t h e h e r b i v o r o u s t o p s h e l l , Tegula f u n e b r a l i s , as b e i n g  12.1  (+ 4.7)% o f t h e dry weight i n t h e female v i s c e r a l mass, and i n t h e male. female of  and  5.5  The  lipid  i n t h e f o o t muscle was  (+ 4 . 2 ) % i n the male.  The  i n t h e ovary.  funebralis  The  lipid  4.3  2.3  (+  was  0.9)%  (+ 2 . 4 ) % i n t h e  d i f f e r e n c e i n the l i p i d  the v i s c e r a l mass between males and females  oocytes  on t h e  p r o b a b l y due  levels  to  l e v e l i n t h e v i s c e r a l mass o f T e g u l a  i s much lower t h a n t h a t i n T. l a m e l l o s a , and  i s perhaps a  r e f l e c t i o n of the d i f f e r e n c e i n d i e t . Glycogen  i n the d i g e s t i v e gland;  The maximum l e v e l o f  glycogen  i n T. l a m e l l o s a d i g e s t i v e gland i s t w i c e as g r e a t as the maximum p o l y s a c c h a r i d e l e v e l o f 3% i n t h e "gonad-free" P a t e l l a v u l g a t a (Blackmore, P. v u l g a t a would be expected  1969).  t i s s u e o f the limpet,  The low p o l y s a c c h a r i d e l e v e l i n  on t h e b a s i s o f t h e f i g u r e s quoted  Emerson (1965) f o r t h e h e r b i v o r o u s l i m p e t s .  by  Blackmore (1969) found  52 l a r g e amounts o f ash i n P. v u l g a t a , r a n g i n g from 15 t o 37% o f t h e t o t a l d r y weight.  He concluded  when t h e a l g a l cover was  t h a t t h e ash content was  highest  a t a minimum, and t h e l i m p e t i n g e s t e d  r e l a t i v e l y more i n o r g a n i c m a t e r i a l a l o n g w i t h t h e f o o d .  In  l a m e l l o s a t h e h i g h e s t ash weights were 8% i n t h e f o o t muscle  T. and  about 7% i n t h e d i g e s t i v e g l a n d . Role o f glycogen that in Patella  i n reproduction:  glycogen l e v e l s  B a r r y and Munday (1959) showed  i n t h e d i g e s t i v e gland were c l o s e l y  c o r r e l a t e d w i t h s e a s o n a l v a r i a t i o n s i n b l o o d g l u c o s e , and t h e y c l u d e d t h a t t h e i n c r e a s e d glycogen There was  a decrease  l e v e l s were p r o b a b l y  of  feeding.  i n glycogen  of  P a t e l l a c o i n c i d i n g w i t h t h e shedding  indicative  level in a l l tissues  o f gametes i n January,,  ever, a f t e r spawning, t h e r e s u l t a n t low l e v e l o f glycogen c o n s t a n t throughout not u t i l i z e  the winter.  I t was  con-  concluded  How-  remained  that Patella did  glycogen t o m a i n t a i n metabolism d u r i n g t h e w i n t e r .  gonad o f P a t e l l a , d u r i n g t h e y e a r , n e v e r c o n t a i n e d a p p r e c i a b l e  The glycogen  r e s e r v e s as compared t o t h e pulmonate, H e l i x , o r t h e o y s t e r , Ostrea gigas.  B a r r y and Munday (1959) and  Blackmore (1969) both  t h a t i n P a t e l l a , t h e major food r e s e r v e i s l i p i d , c  concluded  and t h a t p o l y s a c -  c h a r i d e s a r e not s i g n i f i c a n t as food r e s e r v e s but v a r y w i t h f e e d i n g . G i e s e and  Hart  (1967) found t h a t p o l y s a c c h a r i d e l e v e l s  t u n i c a t a gonad were h i g h e s t when t h e gonad was Blackmore (1969) a l s o found t h i s  i n the oocytes.  s m a l l and  i n P a t e l l a v u l g a t a , and  conclusion that polysaccharide occurred rather than  i n Katherina inactive. came t o t h e  i n the i n t e r s t i t i a l  tissue  As s t a t e d p r e v i o u s l y , t h e amount o f  p o l y s a c c h a r i d e i n marine gastropod  eggs i s low, most o f t h e r e s e r v e s  b e i n g i n t h e form o f p r o t e i n y o l k and l i p i d  yolk.  There i s an  i n c r e a s e i n t h e glycogen l e v e l o f t h e ovary o f T. l a m e l l o s a a f t e r spawning, which l i k e P a t e l l a and K a t h e r i n a , suggest t h a t glycogen Is situated  i n t h e i n t e r s t i t i a l t i s s u e o f t h e ovary.  What r o l e  glycogen has i n t h e ovary i s not known, b u t N i m i t z and Giese u s i n g h i s t o c h e m i c a l t e c h n i q u e s on t h e ovary o f K. t u n i c a t a ,  this (1964), found  t h a t glycogen i n t h e e p i t h e l i u m o f t h e ovary g r a d u a l l y decreased t o i t s lowest v a l u e i n May when spawning t o o k p l a c e . of  glycogen i n t h e e p i t h e l i u m , no glycogen appeared  There was a s i m i l a r decrease o f l i p i d the glycogen, l i p i d  began t o appear  D e s p i t e t h e decrease i n the oocytes.  i n t h e e p i t h e l i u m , but u n l i k e  i n t h e growing  oocytes.  a b l y , t h e glycogen was used i n t h e s y n t h e s i s o f l i p i d yolk.  Presum-  and p r o t e i n  A s i m i l a r s i t u a t i o n o c c u r s i n t h e ovary o f t h e o y s t e r , O s t r e a  gigas.  Glycogen reached a maximum i n A p r i l t h e n began t o d e c l i n e as  the l i p i d  l e v e l increased.  spawning o c c u r r e d .  The l i p i d  l e v e l peaked i n June a f t e r which  I t was p o s t u l a t e d t h a t glycogen i n t h e s t o r a g e  t i s s u e o f t h e gonad was c o n v e r t e d i n t o l i p i d  and t r a n s f e r r e d t o t h e  s e x u a l p r o d u c t s (Masumoto, Masumoto, and H i b i n o , 1934).  Other  c h e m i c a l i n v e s t i g a t i o n s have shown t h a t t h e f o l l i c l e c e l l s  histo-  around  oocytes i n c h i t o n s can r e s o r b n u t r i e n t s from unshed o o c y t e s , s t o r e them, and l a t e r pass them on t o growing 1949;  Selwood, 1970).  oocytes (Gabe and Prenant,  A h i s t o c h e m i c a l study o f T. l a m e l l o s a would  r e v e a l the r o l e o f the various accessory c e l l s  i n t h e movement and  storage of n u t r i e n t s . The  glycogen l e v e l i n t h e ovary o f T. l a m e l l o s a peaked i n  September and A p r i l a t t h e same time as i n t h e d i g e s t i v e g l a n d .  This  suggests t h a t glycogen from i n g e s t e d food i s i n c o r p o r a t e d i n t o t h e ovary as w e l l as i n t o t h e d i g e s t i v e g l a n d , a l t h o u g h n o t t o t h e same extent.  The glycogen l e v e l dropped  i n both ovary and d i g e s t i v e gland  f o l l o w i n g t h e September c o l l e c t i o n and a t t h e same time p r o t e i n lipid  l e v e l s i n t h e ovary Increased ( F i g . 12).  T h i s may  and  have been  due t o t h e u t i l i z a t i o n o f glycogen i n t h e s y n t h e s i s o f p r o t e i n y o l k and l i p i d to  y o l k i n the oocytes.  I f glycogen r e s e r v e s were b e i n g used  s y n t h e s i z e y o l k , i t would be expected t h a t t h e glycogen  would drop and t h e p r o t e i n and l i p i d  level  l e v e l s In t h e ovary would  U n l i k e t h e d i g e s t i v e g l a n d , t h e p r o t e i n and l i p i d  increase.  l e v e l s o f t h e ovary  change i n a p a r a l l e l manner, s i n c e both c o n s t i t u e n t s are major contributors to yolk. C o n s t i t u e n t s o f t h e ovary: index dropped,  At t h e time o f spawning, t h e ovary  but t h e c o r r e s p o n d i n g drop i n l i p i d  i n t h e ovary was  n o t dramatic ( F i g . 12)  i n v e r t e b r a t e s ( G i e s e and H a r t , 1967; 1967;  Blackmore,  1969).  and p r o t e i n  level  as found i n some o t h e r  G i e s e , H a r t , Smith and Cheung,  The r e l a t i v e l y s m a l l b i o c h e m i c a l changes i n  t h e ovary o f T. l a m e l l o s a might be e x p l a i n e d by m o r p h o l o g i c a l evidence. Although t h e egg s i z e data showed l o s s o f mature oocytes a t spawning, i n f a c t some were s t i l l p r e s e n t i n t h e form o f d i s i n t e g r a t i n g gations of y o l k granules.  They were n o t c o n s i d e r e d oocytes as t h e  c r i t e r i o n r e q u i r e d t h e presence o f a n u c l e u s . to  aggre-  d i s i n t e g r a t e and be r e s o r b e d .  Unshed oocytes  appeared  I t has been shown i n t h e c h i t o n ,  S y p h a r o c h i t o n s e p t e n t r i o n e s , t h a t a l l t h e c e l l t y p e s i n the ovary, a p a r t from t h e oocytes and l a t e f o l l i c l e  c e l l s , possess  lysosomes,  and are c a p a b l e o f r e s o r b i n g unshed, mature oocytes and l a t e r p a s s i n g t h e n u t r i e n t s t o new  oocytes (Selwood,  1968).  Some y o l k may  be  d i g e s t e d by t h e i n g e s t i n g gland o f T. l a m e l l o s a , s i t u a t e d between t h e albumen gland and t h e c a p s u l e g l a n d .  S e c t i o n i n g o f t h i s gland  showed t h e presence o f a few y o l k g r a n u l e s .  F r e t t e r (1941) a l s o gave  55 evidence f o r t h e r o l e o f t h e i n g e s t i n g gland i n d i g e s t i n g unused y o l k . The y o l k g r a n u l e s i n a d i s i n t e g r a t i n g oocyte would p r o b a b l y y i e l d s i m i l a r r e s u l t s t o t h e y o l k i n a mature oocyte. ovary i s never c o m p l e t e l y emptied why  The f a c t t h a t t h e  o f mature oocytes p r o b a b l y e x p l a i n s  the b i o c h e m i c a l c o n s t i t u e n t s d i d n o t show dramatic changes. P o l y s a c c h a r i d e as a s t o r a g e p r o d u c t :  In animals which use  p o l y s a c c h a r i d e s as a s t o r a g e p r o d u c t , t h e l e v e l s are much higher than t h e maximum glycogen l e v e l o f 7% i n T. l a m e l l o s a .  The clam,  Tivela  s t u l t o r u m , has from 10 t o 25% glycogen i n t h e non-gonad t i s s u e , 25 t o 50% i n t h e gonads ( G i e s e e t a l . , 1967).  The  abalone,  and  Haliotis  c r a c h e r o d i i , has p o l y s a c c h a r i d e l e v e l s r a n g i n g from 5 t o 25% i n t h e f o o t muscle (Webber and G i e s e , 1969).  In t h e sea u r c h i n , . S t r o n g y l o -  c e n t r o t u s p u r p u r a t u s , t h e gonad appears t o be t h e major s i t e food storage.  Here the maximum glycogen c o n t e n t was  o f h i g h e s t gonad index, and t h e minimum was  of  10% a t t h e time  1% ( G i e s e e t a l . ,  1959a).  Judging" from t h e r e l a t i v e l y low glycogen l e v e l s i n t h e gonad and muscle o f T. l a m e l l o s a , and t h e low l e v e l s o f glycogen i n t h e  foot  digestive  g l a n d , which f l u c t u a t e w i t h f e e d i n g , glycogen i s n o t an important food reserve.  A f t e r i n g e s t i o n I t may  be s t o r e d b r i e f l y t h e n s y n t h e s i z e d  into either l i p i d , . p r o t e i n , or yolk. F u r t h e r evidence f o r t h e r o l e o f glycogen i n T. l a m e l l o s a can o b t a i n e d from t h e e x p e r i m e n t a l data i n T a b l e 3.  be  In t h e t h r e e t i s s u e s  a n a l y s e d , t h e d i f f e r e n c e i n glycogen l e v e l between f e d and s t a r v e d animals was  almost n e g l i g i b l e .  However, the glycogen l e v e l s i n d i g e s t i v e  gland and f o o t muscle from t h e f i e l d 1969, drop  p o p u l a t i o n were lower on January  t h a n a t t h e b e g i n n i n g o f t h e experiment i n glycogen may  on August 31, 1968.  have been due t o e i t h e r c o n v e r s i o n t o l i p i d ,  17,  This or  u t i l i z a t i o n by t h e d e v e l o p i n g o o c y t e s . t h e gonads d i d not develop.  There was  i n t h e s y n t h e s i s o f p r o t e i n or l i p i d did  not d e c r e a s e .  I n t h e f e d and no requirement  I t Is p o s s i b l e t h a t glycogen  M a r t i n (1961) d e s c r i b e d t h e work o f May  pulmonate, H e l i x pomatia.  May  glycogen was was  depleted.  I t was  developing  utilized  by  o n l y drawn upon when  speculated t h a t the r o l e of galactogen  p r i m a r i l y as a r e s e r v e f o r r e p r o d u c t i o n , and because i t was  e a s i l y m o b i l i z e d , t h e s n a i l was  used  (1934) on t h e  found t h a t g l y c o g e n was  t h e s n a i l d u r i n g s t a r v a t i o n , but g a l a c t o g e n was  levels  i s not r e a d i l y  f o r the  groups,  f o r glycogen  y o l k , so t h e glycogen  d u r i n g s t a r v a t i o n , but i s p r e f e r e n t i a l l y used oocytes.  starved  driven to exert i t s e l f  not  in finding  food and t h u s , c a r r i e d through the demanding r e p r o d u c t i v e p r o c e s s . T h i s may i s used ing  be t h e case i n T. l a m e l l o s a except t h a t p r o t e i n and d u r i n g s t a r v a t i o n , and t h a t glycogen  oocytes.  lipid  i s used f o r t h e  develop-  However, more d a t a are r e q u i r e d t o s u b s t a n t i a t e t h i s  hypothesis. L i p i d l e v e l s i n T. l a m e l l o s a ;  The  sites of l i p i d  p r o b a b l y t h e d i g e s t i v e gland and t h e gonad. t h e f o o t muscle was  about 7% throughout  (1966) s t a t e s t h a t t h e l i p i d c h e m i c a l l y appeared  s t o r a g e are  The l e v e l o f l i p i d  t h e y e a r ( F i g - 11).  in  Giese  l e v e l i n crab muscle c e l l s , which h i s t o -  t o s t o r e no l i p i d ,  concluded t h a t a l e v e l o f 5.2%  was  5.2%.  T h e r e f o r e , i t was  represented only s t r u c t u r a l  lipid,  and a n y t h i n g above t h i s p r o b a b l y r e p r e s e n t s s t o r e d l i p i d .  The  level  of  8%,  so  lipid  i n t h e f o o t muscle o f T. l a m e l l o s a d i d not exceed  storage of l i p i d great extent. possess l i p i d  i n t h e f o o t muscle p r o b a b l y does not o c c u r t o  any  S p e c i e s which h i s t o c h e m i c a l l y have been shown t o v a c u o l e s i n t h e f o o t muscle, have much l a r g e r  lipid  values. a lipid  F o r example, t h e f o o t o f t h e c h i t o n , M o p a l i a h i n d s i i , has l e v e l o f 21% and 17% i n g r a v i d and spent a n i m a l s , r e s p e c t i v e l y  ( G i e s e and A r a k i , 1962).  The c h i t o n , C r y p t o c h i t o n  spawns i n A p r i l - M a y , has a l i p i d  stelleri,  which  l e v e l i n t h e f o o t muscle o f 5% o r  l e s s , and i n t h e d i g e s t i v e g l a n d , from 2% t o 10% ( T u c k e r and G i e s e , 1962).  T. l a m e l l o s a ,  on t h e o t h e r hand, has a g r e a t e r  level of l i p i d  i n t h e d i g e s t i v e g l a n d , from 20% t o S8%.  These d i f f e r e n c e s may be  a reflection  i s m a i n l y h e r b i v o r o u s and  therefore,  of their diets.  C. s t e l l e r i  Ingests more p o l y s a c c h a r i d e ,  carnivorous  and t a k e s i n more l i p i d .  s i p u n c u l i d , Phascolosoma g o u l d i i , i n t h e muscle.  whereas T. l a m e l l o s a i s  Prolonged s t a r v a t i o n i n t h e  caused a drop i n a l l l i p i d  In fact, the l e v e l of l i p i d  increased,  except  suggesting  t h a t p r o t e i n o r some o t h e r c o n s t i t u e n t was u t i l i z e d a t a g r e a t e r (Wilber,  1947).  Constituents  o f t h e f o o t muscle;  muscle o f T. l a m e l l o s a the  protein.  ion  ( F i g . 11) f o l l o w e d  The c o n s t i t u e n t  of the foot  which d i d appear t o change s i g n i f i c a n t l y  by a drop i n October.  i n t o t i s s u e p r o t e i n i n t h e f o o t muscle.  The h i g h  level in  at that time.  The  p r o t e i n l e v e l was p r o b a b l y not changing i n response t o a l i p i d i n t h e d i g e s t i v e gland, since the l i p i d  drop i n October corresponded t o an i n c r e a s e  f o o t muscle p r o t e i n a l s o o c c u r r e d  change,  l e v e l remained f a i r l y  stable.  of protein i n the  ovary, which r e s u l t e d from t h e f o r m a t i o n o f p r o t e i n y o l k . in  food  S i m i l a r l y , t h e peak i n A p r i l  was p r o b a b l y due t o t h e resumption o f f e e d i n g  The  was  There was a h i g h p r o t e i n l e v e l i n t h e September c o l l e c t -  September might have been due t o t h e i n c o r p o r a t i o n o f i n g e s t e d  as  rate  This  decrease  d u r i n g t h e time when f e e d i n g  was  reduced, which suggests t h a t p r o t e i n may be e i t h e r m e t a b o l i z e d as a  58 source o f energy, o r u t i l i z e d by t h e o o c y t e s .  The  experimental  data  i n T a b l e 3 show t h a t p r o t e i n i n t h e muscle o f t h e s t a r v e d animals d i d not change but i n t h e f e d group i t i n c r e a s e d s i g n i f i c a n t l y (p <L F u r t h e r , i n t h e two in  experimental  s i z e and no mature oocytes  groups t h e o v a r i e s d i d not  developed.  where t h e o v a r i e s developed l e v e l o f t h e f e d group may evidence,  i t i s suggested  normally.  The  increase  Hence, p r o t e i n was  not r e q u i r e d f o r y o l k p r o d u c t i o n as i n t h e normal f i e l d  .05).  probably  population  increase i n the p r o t e i n  have been a r e s u l t o f f e e d i n g .  From t h i s  t h a t t h e drop i n f o o t muscle p r o t e i n a f t e r  October and t h e peaks i n September and A p r i l were a r e s u l t o f d i f f e r ent r a t e s o f f e e d i n g by T. l a m e l l o s a , r a t h e r t h a n as a r e s u l t t r a n s f e r t o growing Conclusions 25, 1968,  oocytes.  about n u t r i e n t t r a n s f e r :  The  16, 1969  a t a peak.  c o l l e c t i o n , t h e d i g e s t i v e gland  Analyses  o f t h e animals  decreased.  i n c r e a s e d and t h e  I t i s speculated that the increase i n l i p i d  ponse t o a r e d u c t i o n o f food a v a i l a b i l i t y .  was  d i g e s t i v e gland was  synthesis f o r storage.  The  reduced be  l o s s o f weight from t h e  p r o b a b l y due t o glycogen u t i l i z a t i o n f o r l i p i d  y o l k s y n t h e s i s , and l a t e r t o u t i l i z a t i o n o f l i p i d The  glycogen a res-  With t h e onset o f a  food s u p p l y i n g e s t e d n u t r i e n t s and p o l y s a c c h a r i d e r e s e r v e s may into l i p i d  from  from t h e c o l l e c t i o n f o l l o w i n g t h e  d i g e s t i v e gland peak showed t h a t t h e l i p i d had  directed  From  i n s i z e , s u g g e s t i n g t h a t r e s e r v e s were b e i n g u t i l i z e d  t h a t organ.  had  c o l l e c t i o n on October  showed t h a t the d i g e s t i v e gland index was  t h i s data u n t i l February decreased  of  reserves during winter.  s n a i l s presumably l i v e d o f f t h e accumulated l i p i d  and by A p r i l t h e l i p i d was  a t i t s lowest l e v e l .  or  The  during the glycogen  winter,  level  59  had  a l r e a d y begun t o i n c r e a s e by A p r i l as f e e d i n g had resumed.  When  g l y c o g e n reaches a c e r t a i n l e v e l due t o f e e d i n g , some o f i t may be c o n v e r t e d i n t o l i p i d f o r s t o r a g e .  T h i s may  then  e x p l a i n the l a g i n the  l i p i d peak f o l l o w i n g t h e onset o f f e e d i n g i n A p r i l . C o n c l u s i o n s drawn from e x p e r i m e n t a l o b s e r v a t i o n s : e x p e r i m e n t a l work ( T a b l e 3) ovary p l a y i n t h e metabolism demands and s t a r v a t i o n . field  suggest what r o l e s t h e d i g e s t i v e gland and o f t h e s n a i l under normal r e p r o d u c t i v e  The mean index o f t h e d i g e s t i v e glands o f t h e  group decreased s i g n i f i c a n t l y d u r i n g t h e e x p e r i m e n t a l p e r i o d , but  t h e ovary index d i d not change. of  Data from the  t h e ovary was  Thus, i n t h e f i e l d  population the  m a i n t a i n e d , but t h e d i g e s t i v e gland decreased.  c h e m i c a l a n a l y s e s have shown t h a t t h e weight l o s s was of  glycogen, l i p i d ,  at  t h e two  and t o some e x t e n t , p r o t e i n .  Bio-  due t o u t i l i z a t i o n  However, i f one l o o k s  e x p e r i m e n t a l groups, which due t o t h e imposed c o n d i t i o n s d i d  n o t f o l l o w t h e normal r e p r o d u c t i v e p a t t e r n , i t was which remained creased.  the d i g e s t i v e gland  a t a c o n s t a n t s i z e , and r a t h e r t h e ovary s i z e which  I f t h e f e d and s t a r v e d groups  t h e s t a r v e d animals d i d .  de-  are compared i t w i l l be noted  t h e d i g e s t i v e g l a n d index o f both d i d not d e c r e a s e , but t h e ovary of  size  that  index  F u r t h e r , t h e ovary index o f the s t a r v e d group  decreased more t h a n t h e f e d group.  I t seems t h a t under normal  conditions,  r e s e r v e s i n t h e d i g e s t i v e gland are used, but d u r i n g s t a r v a t i o n n u t r i e n t s are  r e s o r b e d from t h e ovary.  formed  However, t h e s t a r v a t i o n experiment  under abnormal c o n d i t i o n s o f l i g h t , temperature,  t h e e f f e c t o f s t a r v a t i o n under normal f i e l d  was  and s a l i n i t y ,  c o n d i t i o n s i s n o t known.  perso In  both t h e e x p e r i m e n t a l groups t h e glycogen l e v e l d i d n o t d e c r e a s e , not even i n t h e s t a r v e d animals.  T h i s s u p p o r t s t h e suggestion;; t h a t t h e p r i m a r y  60 f u n c t i o n o f glycogen i s t o supply t o serve  as a g e n e r a l  the developing  energy r e s e r v e .  came t o t h e c o n c l u s i o n t h a t l i t t l e  Emerson and Duerr (1967) a l s o  glycogen i s used d u r i n g  i n L i t t o r i n a p l a n a x i s , and suggested t h a t anaerobiosis  oocytes r a t h e r t h a n  starvation  i t might be a r e s e r v e f o r  i f s t a r v a t i o n was coupled w i t h d e s s i c a t i o n .  I n summary,  under normal c o n d i t i o n s , t h e data suggest t h a t t h e d i g e s t i v e  gland  supplies n u t r i e n t s t o the developing  body  oocytes and f o r g e n e r a l  maintenance, as shown by t h e r e d u c t i o n the  decrease i n glycogen and l i p i d .  Under abnormal c o n d i t i o n s , when  t h e ovary i s prevented from d e v e l o p i n g , draws on t h e r e s e r v e s Von  o f t h e s i z e o f t h e gland and  i t appears t h a t t h e a n i m a l  o f t h e ovary t o m a i n t a i n i t s body f u n c t i o n s .  Brand, McMahon, and Nolan (1957) showed t h a t s t a r v a t i o n i n  the pulmonate s n a i l , A u s t r a l o r b i s g l a b r a t u s , r e s u l t e d i n a s m a l l l o s s of l i p i d  and p o l y s a c c h a r i d e ,  but t h e m a j o r i t y  o f t h e weight l o s s was  due  to u t i l i z a t i o n of protein.  They d i d n o t measure p r o t e i n  but  on t h e b a s i s o f oxygen consumption data, t h e amount o f oxygen  consumed d i d n o t account f o r t h e s m a l l l o s s i n l i p i d  and  directly,  polysaccharide.  Data f o r a cephalopod i n d i c a t e d t h a t p r o t e i n as w e l l as o t h e r were m e t a b o l i z e d , s i n c e t h e l i p i d  l e v e l remained t h e same w h i l e t h e  animal decreased i n s i z e ( G i e s e , 1966).  Emerson and Duerr (1967), on  t h e o t h e r hand, found t h a t a f t e r s t a r v i n g t h e prosobranch, planaxis, t o t a l l i p i d saccharide  d i d not.  reserves  Littorina  decreased s i g n i f i c a n t l y , w h i l e p r o t e i n and p o l y The e x p e r i m e n t a l data f o r T. l a m e l l o s a  seem t o suggest u t i l i z a t i o n o f l i p i d o f t h e experiment, however, should mental c o n d i t i o n s , t h e s t a r v e d  during starvation.  be c o n s i d e r e d .  do n o t  The c o n d i t i o n s  Under t h e e x p e r i -  animals withdrew n u t r i e n t s from t h e  ovary r a t h e r t h a n from t h e d i g e s t i v e g l a n d .  During t h e winter,  when  61 food i n t a k e i s low, l i p i d In  a review o f l i p i d s  is utilized  from the d i g e s t i v e g l a n d .  i n marine i n v e r t e b r a t e s , Giese (1966) con-  c l u d e d t h a t a l t h o u g h l i p i d has been shown t o be an important r e s e r v e m a t e r i a l , p r o t e i n i s generally present i n a l l t i s s u e s i n a quantity greater than e i t h e r polysaccharide or l i p i d , need a r i s e s .  and may  be used when t h e  G i e s e mentions t h e f o l l o w i n g s p e c i e s as u t i l i z i n g  during starvation:  protein  t h e s i p u n c u l i d , Phascolosoma a g a s s i z i , t h e c h i t o n ,  K a t h e r i n a t u n i c a t a , and t h e shore c r a b , Hemigrapsus nudus.  As men-  t i o n e d p r e v i o u s l y , when an organ decreases i n s i z e , p r o t e i n i s u s u a l l y metabolized along with l i p i d  and glycogen.  The p r o t e i n l e v e l i n t h e  d i g e s t i v e g l a n d o f t h e s t a r v e d T. l a m e l l o s a showed more o f a l o s s t h a n t h e f e d animals. E f f e c t o f E n v i r o n m e n t a l F a c t o r s on Gonad Development Lack o f f o o d :  The ovary index o f t h e s t a r v e d animals  decreased,  p a r t l y because t h e e x p e r i m e n t a l c o n d i t i o n s d i d not a l l o w m a t u r a t i o n of  t h e o o c y t e s , and p a r t l y because  of a l a c k o f food.  The ovary index  of  t h e f e d animals a l s o d e c l i n e d but not t h e same e x t e n t .  Hence,  a v a i l a b i l i t y o f f o o d a l l o w e d t h e ovary t o m a i n t a i n i t s s i z e , but t h e p h y s i c a l c o n d i t i o n s prevented m a t u r a t i o n o f t h e o o c y t e s .  Mature  oocytes which were p r e s e n t a t t h e b e g i n n i n g were i n t h e p r o c e s s o f being resorbed. presumably  The  s i z e o f t h e ovary o f t h e s t a r v e d animals was  less,  due t o t h e g r e a t e r demand on t h e r e s e r v e s i n t h e ovary.  S a s t r y (1966) found t h a t s t a r v a t i o n o f s c a l l o p s d u r i n g t h e p e r i o d gonad growth r e s u l t e d i f t h e animals had  i n an a b s o r p t i o n o f oogonia and o o c y t e s , but  a l r e a d y accumulated  gametes, r e g a r d l e s s .  of  gonad r e s e r v e s , t h e y r e l e a s e d  The pulmonate, Lymnaea s t a g n a l i s , i f s t a r v e d  for  s i x weeks, m a i n t a i n e d a l l stages o f oogenesis and  i n t h e o v o t e s t i s ; however t h e number o f sex c e l l s t h e o v o t e s t i s was  reduced.  spermatogenesis  and t h e s i z e o f  A l a r g e number o f mature oocytes degen-  e r a t e d and were r e s o r b e d by t h e nurse c e l l s , but d e v e l o p i n g oocytes d i d n o t seem t o be a f f e c t e d ( J o o s s e , Boer, and C o r n e l i s s e , Temperature:  1968).  In T. l a m e l l o s a , m a i n t a i n e d under summer c o n d i t i o n s  o f l i g h t , temperature,  and s a l i n i t y , t h e sex c e l l s p r o l i f e r a t e d more  i n f e d than i n s t a r v e d animals, but i n n e i t h e r case d i d t h e oocytes mature.  Thus, i t c o u l d be s a i d t h a t the c o n d i t i o n f o r gonad matur-  a t i o n i s a f a c t o r o t h e r t h a n food s u p p l y . o f l i g h t , temperature, i t was  and s a l i n i t y was  applied i n the  n o t p o s s i b l e t o conclude which was  S a s t r y (1968) showed t h a t t h e r e was and temperature Aequipecten  S i n c e o n l y one  of the  scallop,  w i t h food seemed t o  When temperatures  were below optimum,  t h e r e s e r v e s from t h e i n g e s t e d f o o d seemed t o accumulate d i g e s t i v e gland t h a n i n t h e gonad.  factor.  a r e l a t i o n s h i p between food s u p p l y  Optimum temperatures  i n i t i a t e gonad development.  experiment,  the c o n t r o l l i n g  and t h e r e p r o d u c t i v e a c t i v i t y  irradians.  combination  more In t h e  In t h e p r e s e n t study, e x p e r i m e n t a l  animals i n temperatures h i g h e r t h a n t h e f i e l d , m a i n t a i n e d t h e gland s i z e , and u t i l i z e d was to  r e s e r v e s from t h e gonad.  f a r above t h e optimum, t h e r e s e r v e s o f A. accumulate  utilization,  i n t h e gonad e i t h e r because  I f the  digestive  temperature  i r r a d i a n s d i d n o t seem  of increased metabolic  f a i l u r e t o r e g u l a t e t h e s y n t h e t i c p r o c e s s e s o f t h e grow-  ing  o o c y t e s , o r death o f t h e s c a l l o p s .  Furthermore,  may  develop below t h e optimum temperature  o o c y t e development d i d not seem t o o c c u r . T. l a m e l l o s a , but i n t h e o p p o s i t e way.  although  when food was T h i s may  oogonia  supplied,  be t h e case i n  These s n a i l s breed a t t h e  lowest temperatures may  o f t h e y e a r ; t h e r e f o r e an optimum low  be r e q u i r e d t o a l l o w t h e oocytes t o mature.  Two  temperature  species of  b a r n a c l e s , Balanus b a l a n o i d e s and B. b a l a n u s , which breed i n November and F e b r u a r y , r e s p e c t i v e l y , d i d not breed a t temperatures 18°C but d i d i n t h e range o f 3 t o 10°C  ( C r i s p , 1957).  o f 14 t o  The dog whelk,  T h a i s l a p i l l u s , under a r t i f i c i a l  laboratory conditions, required  to  i n f o r m a t i o n , however was  s t i m u l a t e egg d e p o s i t i o n .  t h e e f f e c t o f temperature (Largen,  No  on gametogenesis  9°C  g i v e n about  or oocyte m a t u r a t i o n  1967).  Day l e n g t h : breeding c y c l e s .  Day l e n g t h has been i m p l i c a t e d  Bo.olootian (1963) m a i n t a i n e d male p u r p l e sea  u r c h i n s a t a c o n s t a n t temperature On a 14-hour day,  i n the c o n t r o l o f  initial  o f 15°C  '  and v a r i e d t h e d a y - l e n g t h .  development o f g o n i a l c e l l s began but  no mature sperm r e s u l t e d .  On a 6-hour day, g o n i a l c e l l s were reduced  i n number as t h e y developed  i n t o spermatocytes.  u r c h i n used  The p u r p l e sea  i n t h e study n o r m a l l y reproduces i n t h e w i n t e r .  (1963) performed  s i m i l a r experiments  Barnes  on w i n t e r - b r e e d i n g B. b a l a n u s .  Constant i l l u m i n a t i o n i n h i b i t e d b r e e d i n g , e s p e c i a l l y t h e l a t e r stages of  development.  p e r day was  A p e r i o d o f 4 t o 6 weeks a t l e s s t h a n 12 hours  light  r e q u i r e d f o r m a t u r a t i o n o f gametes.  Webber and G i e s e (1969) a n a l y s e d b r e e d i n g data f o r H a l i o t i s c r a c h e r o d i i and K a t h e r i n a t u n i c a t a c o l l e c t e d over s e v e r a l y e a r s , and concluded t h a t temperature gonad growth. in  d i d not a c t as an exogenous c o n t r o l o f  P h o t o p e r i o d d i d not show a c o r r e l a t i o n w i t h t h e i n c r e a s e  gonad s i z e , but i t was  suggested t h a t gametogenesis  by day l e n g t h s o f g r e a t e r t h a n a p p r o x i m a t e l y 12 hours.  was  initiated  I t was  empha-  s i z e d t h a t mere c o r r e l a t i o n o f e n v i r o n m e n t a l changes cannot be  viewed  64 as c o n c l u s i v e , and  data from c o n t r o l l e d experiments should  to establish causal factors. a l s o suggested as an  The  important  r o l e o f t h e endocrine  be  obtained  system  was  c o n s i d e r a t i o n f o r t h e f u t u r e study  of  mollusk reproductive c y c l e s . Salinity:  S a l i n i t y has  not g e n e r a l l y been c o n s i d e r e d  as a  stimulus  f o r r e p r o d u c t i o n , but i n s i t u a t i o n s such as Brockton P o i n t , where r e l a t i v e l y l a r g e s a l i n i t y f l u c t u a t i o n s t a k e p l a c e (17 t o 29°/oo), i t should not be d i s c o u n t e d .  I t c o u l d be argued t h a t i n r e g i o n s  open c o a s t temperature and  s a l i n i t y f l u c t u a t i o n s are v e r y low,  breeding  occurs  at r e g u l a r t i m e s .  I t may  on  the  yet  be t h a t p o p u l a t i o n s  of a  s p e c i e s i n d i f f e r e n t l o c a l i t i e s adapt t o t h a t p a r t i c u l a r environment, and  u t i l i z e t h e most obvious r e g u l a r changes i n t h e environment, t o  time t h e b r e e d i n g  cycle.  s t u d i e d by Webber and  F o r example, t h e p o p u l a t i o n o f H. c r a c h e r o d i i ,  Giese  (1969), encountered o n l y s m a l l f l u c t u a -  t i o n s i n temperature (range: breed  regularly.  There was  5°C) and  s a l i n i t y , y e t appeared t o  a c o r r e l a t i o n between day l e n g t h  i n i t i a t i o n o f gametogenesis, however.  As mentioned i n t h e  paragraph, endogenous rhythms i n t h e endocrine important The  previous probably  i n t h e r e g u l a t i o n of* t h e b r e e d i n g c y c l e . results cited  t e b r a t e s , g i v e evidence light,  system a r e  and  i n the previous  few  paragraphs f o r o t h e r  f o r t h e r o l e o f f e e d i n g , temperature,  i n the c o n t r o l of breeding  cycles.  inver-  and  Until controlled experi-  ments, u s i n g v a r i o u s combinations o f temperature, s a l i n i t y and  light  can be performed no c o n c l u s i o n s can be drawn about t h e f a c t o r s c o n t r o l l i n g reproduction  in Thaislamellosa.  Suggestions f o r F u r t h e r The  data c o l l e c t e d  Study i n t h i s study p r o v i d e a b a s i s f o r f u r t h e r  65 s t u d i e s i n t o T. l a m e l l o s a r e p r o d u c t i o n .  F o r example, h i s t o c h e m i c a l  data would e l u c i d a t e t h e d i s t r i b u t i o n o f n u t r i e n t s w i t h i n t h e t i s s u e s , which so f a r can o n l y be s p e c u l a t e d .  The t e c h n i q u e s  of radioactive  l a b e l l i n g c o u l d be employed t o show t h e s i t e s o f s y n t h e t i c a c t i v i t y and t h e sources  o f p r e c u r s o r s i n r e l a t i o n t o food.  " T r a c e r experiments  are needed t o determine t h e movement o f n u t r i e n t i n marine i n v e r t e b r a t e s " ( G i e s e , 1966, p. 286).  T h i s method was a l s o suggested  by Barnes e t a l .  (1963) as a means o f t r a c i n g t h e p a r t i t i o n o f t h e v a r i o u s m a t e r i a l s d u r i n g growth and development o f t h e gonads.  A more a c c u r a t e d e t e r -  m i n a t i o n o f f e e d i n g times d u r i n g t h e year c o u l d be o b t a i n e d by measuring t h e b l o o d g l u c o s e l e v e l s i n t h e manner d e s c r i b e d by B a r r y and Munday A l s o , biochemical analyses  (1959).  o f t h e eggs would h e l p t o e x p l a i n f u r t h e r t h e  gross changes i n t h e gonad.  Very l i t t l e  endocrine  and i t s r o l e i n t h e c o n t r o l o f r e p r o d u c t i o n  system o f m o l l u s c s  work has been done on t h e  (Boer, Douma, and Koksma, 1968; Webber and G i e s e , 1969). the e f f e c t s o f environmental  A study o f  f a c t o r s on r e p r o d u c t i o n , u s i n g c o n t r o l l e d  c o n d i t i o n s , would a l s o p r o v i d e a good o p p o r t u n i t y t o l o o k a t t h e r o l e o f n e u r o s e c r e t i o n i n c o n t r o l l i n g t h e b r e e d i n g cycles.S e v e r a l authors have p o i n t e d out t h e need f o r f u r t h e r i n v e s t i g a t i o n i n t o s e a s o n a l b i o c h e m i c a l changes, and t h e r e l a t i o n s h i p s between nut r i t i o n and r e p r o d u c t i o n i n m o l l u s k s  (Barnes  e t a l . , 1963; A n s e l l and  Lander, 1967; Emerson, 1965, 1967; G i e s e , 1966).  The p r e s e n t work has  attempted t o answer some o f t h e s e q u e s t i o n s with r e g a r d t o t h e p r o s o branch,  Thais lamellosa.  The data show t h e g e n e r a l s i m i l a r i t i e s between  T. l a m e l l o s a and o t h e r prosobranchs uents  and r e p r o d u c t i o n .  with regard t o biochemical  constit-  66 SUMMARY  1.  A p o p u l a t i o n o f T h a i s l a m e l l o s a , from Brockton  Vancouver, B r i t i s h Columbia, was sampled a p p r o x i m a t e l y J u l y 28, 1968, u n t i l August 25, 1969.  Point, monthly from  A study was made o f t h e b i o -  c h e m i c a l changes a s s o c i a t e d w i t h t h e r e p r o d u c t i v e c y c l e . 2.  From t h e t h r e e t i s s u e s , d i g e s t i v e g l a n d , f o o t muscle and  gonad; per cent p r o t e i n , glycogen,  lipid,  As w e l l , gonad and d i g e s t i v e gland  i n d i c e s and h i s t o l o g i c a l s e c t i o n s  of  and ash were o b t a i n e d .  t h e gonad and d i g e s t i v e gland were o b t a i n e d . 3.  The r e p r o d u c t i v e h a b i t s o f T. l a m e l l o s a and some o f t h e  p r o p e r t i e s o f t h e eggs were d i s c u s s e d i n r e l a t i o n t o l e n g t h o f b r e e d i n g season,  gametogenesis, developmental  s u l e , and t i m i n g o f t h e b r e e d i n g 4.  Estimates  time w i t h i n t h e cap-  cycle.  o f s t o r a g e granules  i n t h e d i g e s t i v e gland d i v e r -  t i c u l a e were used t o determine p e r i o d s o f f e e d i n g a c t i v i t y . was a f e e d i n g peak i n A p r i l and another  i n August,  During t h e w i n t e r  months u n t i l spawning i n March, t h e r e was a decrease of  There  i n t h e amount  feeding. 5.  In the f a l l  gland as l i p i d , glycogen 6.  i n g e s t e d food  i s accumulated  but i n p e r i o d s o f a c t i v e f e e d i n g , A p r i l and August,  i s h i g h e s t and l i p i d I t was concluded  i s a t a minimum.  t h a t glycogen  i s n o t used as an energy  r e s e r v e , but i s e i t h e r converted t o l i p i d used by t h e growing oocytes 7.  i n the digestive  i n t h e d i g e s t i v e gland or  f o r yolk production.  The f o o t muscle does n o t s t o r e l i p i d  o r glycogen t o any  a p p r e c i a b l e e x t e n t , b u t t h e p r o t e i n appears t o f l u c t u a t e i n response to  food i n t a k e .  The p r o t e i n l e v e l i n c r e a s e d d u r i n g maximum f e e d i n g  and decreased when f e e d i n g was 8.  In t h e f i e l d  low.  p o p u l a t i o n , t h e d i g e s t i v e gland index decreased  as r e s e r v e s were used, but t h e s i z e o f t h e ovary was about October u n t i l 9.  maintained  from  spawning i n March.  Under t h e e x p e r i m e n t a l c o n d i t i o n s , t h e d i g e s t i v e gland o f  both f e d and s t a r v e d animals maintained i t s s i z e , w h i l e the ovary decreased  in size.  The s t a r v e d animals withdrew more m a t e r i a l from  t h e ovary t h a n t h e f e d animals. 10. to  Mature oocytes i n t h e ovary o f t h e s t a r v e d animals  appeared  be r e s o r b e d , l e a v i n g t h e s m a l l e r oogonia, i n t h e range o f 0 t o  microns.  200  L i k e w i s e , a f t e r spawning, unshed mature oocytes were  resorbed. 11.  The r o l e o f temperature,  r e p r o d u c t i o n was  light,  and s a l i n i t y ,  i n timing  d i s c u s s e d , but c o n c l u s i o n s c o u l d not be drawn based  on c o r r e l a t i o n s w i t h f i e l d  data.  68 LITERATURE CITED  Ahmed, M., and A. K. Sparks, 1970. A note on t h e chromosome number and i n t e r r e l a t i o n s h i p s i n t h e marine gastropod genus T h a i s o f t h e U n i t e d S t a t e s P a c i f i c c o a s t . V e l i g e r , 12:293-294. A n s e l l , A. D., and K. F. Lander, 1967. 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