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Studies of the blood cells and tunic of the ascidian, Halocynthia Aurantium (Pallas) Smith, Michael Joseph 1969

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STUDIES OF THE BLOOD CELLS AND TUNIC OF THE ASCIDIAN, HALOCYNTHIA AURANTIUM (PALLAS) by MICHAEL JOSEPH SMITH B . S c , S t . Mary's C o l l e g e o f C a l i f o r n i a , 1963 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department °f Zoology We accept t h i s t h e s i s as conforming to the r e q u i r e d s tandard. THE UNIVERSITY OF BRITISH COLUMBIA AUGUST 1969 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 r e q u i r e m e n t s f o r an advanced d e g r e e a t the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and S t u d y . I f u r t h e r a g r e e 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 c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s thes , is 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 w r i t t e n p e r m i s s i o n . Department o f The U n i v e r s i t y o f B r i t i s h Co lumbia Vancouver 8, Canada Date 7 i ABSTRACT The morphology and histochemistry of the blood c e l l s and tunic of the ascidian Halocynthia aurantium were studied. The nature of the tunic was investigated by both chemical and biochemical means. Quantitative studies of the blood of Halocynthia display ten blood c e l l types, four of which are concentrated in tunic in higher concentrations than i n blood. Of these four, two, including an iron bearing c e l l , d i s p l a y d i s c r e t e stable aggregation areas i n the body of the tunic, and a t h i r d i s a phagocyte. Histochemical examination of the tunic and blood c e l l s reveals that the tunic f i b e r s and epidermis s t a i n for acid mucopolysaccharide and protein. The spinous processes on the surface of the tunic do not s t a i n l i k e the epidermis or f i b e r s , but do d i s p l a y some s i m i l a r i t y to the blood c e l l type which aggregates i n t h e i r proximity. There i s a coincidence of absorption spectra of methanol extracts of blood c e l l s and tunic, but no p a r t i c u l a r blood c e l l has been indicated as the pigment c e l l . The blood c e l l s , in the tunic, do not display s t a i n i n g properties which would indicate that they are contributing'to the carbohydrate component. Morphological c h a r a c t e r i s t i c s and histochemical properties of epidermis indicate that i t i s the major tunic secreting t i s s u e . The h i s t o -genetic r e l a t i o n s h i p of various blood c e l l s i s discussed on the basis of analogous histochemistry and quantitative hematology. The f u n c t i o n o f p a r t i c u l a r b l o o d c e l l types i n the t u n i c i s suggested by the d i s c r e t e p o s i t i o n a l r e l a t i o n s h i p s o f c e l l s and the morphology of the t u n i c . The chemical composition o f t u n i c i s approximately 50% p r o t e i n and 50% carbohydrate. Amino a c i d a n a l y s i s o f t u n i c b e f o r e and a f t e r p r o t e o l y t i c enzyme treatment shows t h a t the p r o t e i n does not have the c h a r a c t e r i s t i c s o f common c o n n e c t i v e t i s s u e p r o t e i n s such as c o l l a g e n , e l a s t i n , or others, and t h a t the p r o t e i n - p o l y s a c c h a r i d e l i n k p r o b a b l y i n v o l v e s glucosamine and s e r i n e . F i v e p e r cent of the d r y weight of t u n i c i s hexo-samine w i t h both galactosamine and glucosamine p r e s e n t i n a r a t i o o f 1 t o 4. The carbohydrate component of the t u n i c r e l e a s e s 75% of i t s weight as g l u c o s e upon a c i d h y d r o l y s i s . T h i s carbohydrate c o n s i s t e n t l y d i s p l a y s a n e g a t i v e P.A.S. r e a c t i o n and i s . r e s i s t a n t to s e v e r a l c e l l u l o s e d i s p e r s i n g r e a g e n t s . The elemental composition o f the carbohydrate r e v e a l s a hydrogen content which i s too low and an oxygen content which i s too h i g h to i n d i c a t e c e l l u l o s e . Tunic, pronase t r e a t e d t u n i c , and t u n i c carbohydrate were submitted to c e l l u l a s e , c h i t i n a s e , and h y a l u r o n i d a s e d i g e s t i o n . None o f these m a t e r i a l s are s u b j e c t to d e g r a d a t i o n by c h i t i n a s e o r h y a l u r o n i d a s e . T u n i c i s r e f r a c t o r y t o c e l l u l a s e . Pronase t r e a t e d t u n i c w i l l r e l e a s e 20% o f i t s weight as glucose upon i n c u b a t i o n w i t h c e l l u l a s e . T u n i c carbohydrate component w i l l r e l e a s e 55% o f i t s weight as g l u c o s e upon c e l l u l a s e treatment,, although chemical p r o p e r t i e s i n d i c a t e t h a t i t may be more complex than c e l l u l o s e . i i i TABLE OF CONTENTS. Page L i s t of Tables iv L i s t of Figures... o v i Acknowledg ements v i i i Introduction ' 1 Materials and Methods 4 1. General 4 2. H i s t o l o g i c a l Methods 6 3. Histochemical Methods 8 4. Hematological Methods 9 5. Tunic Structure and Injury Studies 10 6. Carbon Phagocytosis • 14 7. Chemical Analyses 14 8. Enzymatic Analyses 21 Results ^ 26 1. Wet versus /Dry Weight 26 2. The Cytology of the Blood 26 3. Hematology 30 4. Morphology of the Tunic 34 5. Blood C e l l s i n the Tunic 47 6. Blood C e l l Function in the Tunic 52 7. Histochemistry of the Blood C e l l s 61 8. Histochemistry of the Tunic 66 9. Enzymatic Analyses of Tunic and Tunicin 68 10. Chemical Analyses of Tunic and Tunicin 72 Discussion 86 Summary 132 L i t e r a t u r e Cited ,135 iv LIST OF TABLES Table Page 1 The s i g n i f i c a n c e of differences of mean per cent of stem c e l l s in blood between 20 gram wet weight groups of H. aurantium 36 2 The s i g n i f i c a n c e of differences of mean per cent of stem c e l l s with a c i d o p h i l i c vacuoles and granules in blood between 20 gram wet weight groups of H. aurantium 37 3 The s i g n i f i c a n c e of differences of mean per cent of 'vacuolar' c e l l s in blood between 20 gram wet weight groups of H. aurantium 38 4 The s i g n i f i c a n c e of differences of mean per cent of mature morula c e l l s in blood between 20 gram wet weight groups of H. aurantium 39 5 The s i g n i f i c a n c e of differences of mean per cent of dispersed v e s i c u l a r c e l l s in blood between 20 gram wet weight groups of H. aurantium 40 6 The s i g n i f i c a n c e of differences of mean per cent of hyaline amoebocytes in blood between 20 gram wet weight groups of H.aurantium 41 7 The r e l a t i v e concentration of blood c e l l types in the blood of H. aurantium as mean per cent 42 8 The concentration of blood c e l l s per cubic millimeter in blood and tunic 49 9 The s i g n i f i c a n c e of differences of mean per cent of hyaline amoebocytes with carbon p a r t i c l e s as a function of time a f t e r carbon i n j e c t i o n 54 10 The s i g n i f i c a n c e of differences i n number of mature morula c e l l s in tunic as a function of time a f t e r injury 56 11 The s i g n i f i c a n c e of differences in number of dispersed v e s i c u l a r c e l l s in tunic as a function of time a f t e r injury 57 12 The s i g n i f i c a n c e l e v e l s of differences in mean concentration of dispersed v e s i c u l a r c e l l s and matura moruls c e l l s per cubic millimeter between the inner and outer h a l f of the tunic as a function of time a f t e r injury 59 V LIST OF TABLES Table Page 13 The histochemical reactions of the hyaline amoebocyte and granular amoebocyte in the blood of H. aurantium 64 14 The histochemical reactions of the dispersed v e s i c u l a r c e l l and mature morula c e l l in blood and tunic 65 15 The histochemical reactions of the three tunic components 69 16 The e f f e c t s of various experimental treatments on the structure and A l c i a n Blue-P.A.S. r e a c t i v i t y of tunic f i b e r s and spines 73 17 The r e l a t i v e loss in dry weight of tunic upon various treatments 75 18 The d i s t r i b u t i o n of iron in various tissues of H. aurantium 76 19 The composition of c e l l u l o s e , tunic, and t u n i c i n in terms of per cent of carbon, hydrogen, oxygen, nitrogen, and s u l f u r 79 20 The molar r a t i o s (as per cent) of the eighteen amino acids in tunic hydrolysates before and af t e r 48 hours incubation with pronase 85 v i LIST OF FIGURES F i g u r e Page 1 The b l o o d c e l l s o f H a l o c y n t h i a aurantium 31 2 The per cent d i s t r i b u t i o n o f b l o o d c e l l types i n b l o o d as a f u n c t i o n o f 20 gram whole animal wet weight groups 33 3 The per cent d i s t r i b u t i o n o f h y a l i n e amoebocytes i n b l o o d as a f u n c t i o n o f 20 gram whole animal wet weight group 35 4 The t u n i c o f H a l o c y n t h i a aurantium 44 5 The number o f laminae, o r f i b r o u s l a y e r s , i n the t u n i c as a f u n c t i o n o f whole animal wet weight i n grams 45 .6 The s t o l o n and i n j u r e d t u n i c o f H a l o c y n t h i a aurantium 48 7 The c o n c e n t r a t i o n , i n thousands o f c e l l s , p e r c u b i c m i l l i m e t e r o f mature morula and d i s p e r s e d v e s i c u l a r c e l l s as a f u n c t i o n o f p o s i t i o n i n the t u n i c 51 8 The per cent of h y a l i n e amoebocytes w i t h carbon p a r t i c l e s as a f u n c t i o n o f time a f t e r i n j e c t i o n i n t o the t u n i c 53 9 The c o n c e n t r a t i o n o f mature morula c e l l s , i n c l u d i n g t u n i c v e s i c u l a r c e l l s , and d i s p e r s e d v e s i c u l a r c e l l s (x 10^) per c u b i c m i l l i m e t e r o f t u n i c as a f u n c t i o n o f day a f t e r i n j u r y 55 10 The e f f e c t o f p h o s p h o r i c a c i d treatment on the y i e l d o f r e d u c i n g sugar from t u n i c i n and pronase d i g e s t e d t u n i c upon c e l l u l a s e d i g e s t i o n 71 11 The a b s o r p t i o n s p e c t r a o f methanol e x t r a c t s o f b l o o d c e l l s and t u n i c o f H. aurantium 78 12 The y i e l d o f r e d u c i n g sugar from t u n i c i n as a f u n c t i o n o f time of h y d r o l y s i s i n 7 2% H 2 S 0 4 81 v i i LIST OF FIGURES F i g u r e Page 13 The e l u t i o n p a t t e r n s o f glucosamine and galactosamine from a 48 cm. column o f Dowex 50W X-8 c a t i o n exchange r e s i n 83 ACKNOWLEDGEMENTS I wish to thank Dr. P.A. Dehnel for h i s continuing support and encouragement throughout t h i s study. I would l i k e also to thank Drs. C u l l i n g , Finnegan, Perks and P h i l l i p s for t h e i r advice and c r i t i c i s m . I must thank Miss E. McGowan for her assistance with the amino acid analyses, Miss M. Young for her ear l y assistance with the histology and histochemistry, and Mrs. J . Englehardt, Miss J. Webber, Miss M. Baldwin, Mr. V. Swiatkewicz, and Mr. D. Larrivee for translations from French. I am indebted to Mr. H. Burton for photographic plates, and Mrs. M. Douglas for preparing the figures. I extend my gratitude to the National Research Council of Canada for a Predoctoral Studentship and the family of K i t Malkin for the K i t Malkin Scholarship. 1 INTRODUCTION Two unique p r o p e r t i e s o f a s c i d i a n s have e l i c i t e d i n t e r e s t o f z o o l o g i s t s f o r over a ce n t u r y . These are the presence o f a c e l l u l o s e - l i k e polymer i n the t u n i c o f these animals and the a b i l i t y o f a s c i d i a n s t o c o n c e n t r a t e t r a n s i t i o n m e t a l s . The presence o f c e l l u l o s e i n the a s c i d i a n , P h a l l u s i a  mammillata, f i r s t was suggested by C. Schmidt i n 1845 ( c i t e d i n S e e l i g e r and Hartmeyer 1911) . The nature o f the carbohydrate i n the t u n i c o f C y n t h i a p a p i l l o s a was i n v e s t i g a t e d by B e r t h e l o t i n 1858 ( i b i d . ) and he proposed t h a t t h e r e were d i f f e r e n c e s between the a s c i d i a n carbohydrate and the c e l l u l o s e o f p l a n t s . As a r e s u l t he co i n e d the word " t u n i c i n " t o d i f f e r e n t i a t e the a s c i d i a n p r o d u c t from p l a n t c e l l u l o s e . W i n t e r s t e i n (1894) agreed t h a t some monosaccharide, o t h e r than glucose, might be p r e s e n t i n the p o l y s a c c h a r i d e m a t e r i a l . Consequently, a number o f workers i n v e s t i g a t e d the nature o f t u n i c i n ; a c e t y l a t e d p r o d u c t s o f t u n i c i n correspond t o a c e t y l a t e d c e l l u l o s e p r o d u c t s (Abderhalden and Zemplen, 1911), the paper chromotography o f a c i d h y d r o l y s a t e s o f t u n i c i n y i e l d spots which have Rf v a l u e s c o r r e s p o n d i n g to c e l l u l o s e h y d r o l y s i s p r o d u c t s (Tsuchiya and Suzuki, 1952), and X-ray d i f f r a c t i o n p a t t e r n s o f the carbo-h y d r a t e d e r i v e d from P h a l l u s i a n i g r a resemble those o f p l a n t c e l l u l o s e (Spence and Richards, 1940). B e f o r e p u r i f i c a t i o n procedures the t u n i c has a much more complicated chemical and h i s t o c h e m i c a l s t r u c t u r e (Vagas, 1962; Godeaux, 1963). The u n t r e a t e d t u n i c o f A s c i d i e l l a a s persa has 2 a c l o s e l y associated protein which resembles r e t i c u l i n (Hall and Saxl, 1961) and hexosamines have been reported in the tunics of Pyura s t o l o n i f e r a (Endean, 1955) and P h a l l u s i a  mammillata (Endean, 1961). In 1911, Henze f i r s t reported the concentration of vanadium in an ascidian. Subsequently, a number of species have been investigated for the presence of vanadium (summarized i n Bertrand, 1950; Webb, 1956;and Cierescko, 1963), which i s p r i m a r i l y concentrated in a blood c e l l termed the vanadocyte (Webb, 1939)„ I t has been noted, however, that not a l l species of tunicate concentrated vanadium, and iron has been reported i n Pyura s t o l o n i f e r a (Endean, 1953). Again the highest concentration of metals was found to be in the blood c e l l s and the p a r t i c u l a r c e l l type with iron was termed a ferrocyte (Endean, 1955a). Other ascidians concentrate niobium ( C a r l i s l e , 1958), and at l e a s t one species concentrates three t r a n s i t i o n metals: titanium, which is found in the highest concentration, vanadium, and chromium, which i s the lowest concentration (Levine, 1962) . Henze (1932) suggested that the presence of vanadium i n the blood c e l l s might be related to the formation of tunic c e l l u l o s e . The blood c e l l s of the ascidian consistently are found i n the tunic ( B e r r i l l , 1950) and among these c e l l s either vanadocytes (Hecht, 1918; Endean, 1961) or ferrocytes (Endean, 1955b) may be i d e n t i f i e d . There are always, however, a v a r i e t y of other blood c e l l types present i n the tunic ( B e r r i l l , 1950). It has been reported that vanadocytes and ferrocytes are 3 r e s p o n s i b l e f o r t u n i c i n s e c r e t i o n (Endean, 1955b, and 1961). In o p p o s i t i o n to t h i s view, i t has been s t a t e d t h a t the e pidermis i s the t u n i c forming t i s s u e (Deck, Hay, and Revel, 1966). C l a s s i c a l l i t e r a t u r e i n d i c a t e s t h a t both epidermis and b l o o d c e l l s p a r t i c i p a t e i n t u n i c s e c r e t i o n . In s t u d i e s o f a s c i d i a n b l o o d c e l l s and t h e i r involvement i n the t u n i c t h e r e has been l i t t l e q u a n t i t a t i v e i n f o r m a t i o n and l e s s s t a t i s t i c a l treatment t h e r e o f . In t h i s study, the metal content, b l o o d c e l l s , and t u n i c o f H a l o c y n t h i a aurantium are i n v e s t i g a t e d i n d e t a i l . P r i m a r i l y , the morphology o f b l o o d c e l l s and t u n i c are d e s c r i b e d . The involvement o f p a r t i c u l a r b l o o d c e l l types i n the t u n i c i s demonstrated by s t a t i s t i c a l comparisons o f d i f f e r e n c e s i n c e l l c o n c e n t r a t i o n o f b l o o d versus t u n i c . F u r t h e r , a g g r e g a t i o n areas o f p a r t i c u l a r b l o o d c e l l types i n the t u n i c are e s t a b l i s h e d s t a t i s t i c a l l y . The f u n c t i o n of b l o o d c e l l s i n the t u n i c i s i n d i c a t e d by p h a g o c t y o s i s s t u d i e s and the c o n c e n t r a t i o n o f p a r t i c u l a r c e l l types i n i n j u r e d t u n i c and i n the r a p i d l y growing t u n i c o f s t o l o n s . H i s t o c h e m i c a l evidence shows t h a t the t u n i c has the p r o p e r t i e s o f an a c i d mucopoly-s a c c h a r i d e and t h a t the epidermis i s the major t u n i c s e c r e t -i n g t i s s u e . In g e n e r a l , the t u n i c does not d i s p l a y p r o p e r t i e s which would i n d i c a t e t h a t i t i s c e l l u l o s e . Chemical and b i o -c hemical s t u d i e s p r o v i d e d a t a f o r the d i s c u s s i o n o f the nature o f the p r o t e i n - p o l y s a c c h a r i d e l i n k a g e . A h y p o t h e s i s f o r the d i f f e r e n t i a t i o n o f metal b e a r i n g b l o o d c e l l types i s proposed based on h i s t o c h e m i c a l d a t a and changes i n r e l a t i v e b l o o d c e l l numbers as a f u n c t i o n o f whole animal wet weight. 4 MATERIAL AND METHODS 1. G e n e r a l : The t u n i c a t e , H a l o c y n t h i a aurantium ( P a l l a s ) , was i d e n t i f i e d by the author based on the work o f Van Name (1945). The animals were c o l l e c t e d throughout the year from the f o l l o w -i n g l o c a t i o n s i n Howe Sound, B r i t i s h Columbia: W h y t e c l i f f Park, Copper Cove, Horseshoe Bay, B r i t a n i a Beach and Sunset Beach. The a s c i d i a n s were c o l l e c t e d by scuba d i v i n g a t depths o f 10 -20 meters. A s c i d i a n s were h e l d i n c o n s t a n t l y a e r a t e d sea water i n e i t h e r g l a s s a q u a r i a , 30 cm. x 60 cm. x 30 cm., on water t a b l e s , o r i n wooden boxes, 46 cm. x 69 cm. x 36 cm., i n a c o n t r o l l e d environmental room. The temperature o f the sea water i n the a q u a r i a on the water t a b l e s v a r i e d from 10°C i n the w i n t e r to 16°C i n the summer. The temperature i n the e n v i r o n -mental room was maintained a t 10°C. A f t e r c o l l e c t i o n , animals were k e p t i n the l a b o r a t o r y f o r a t l e a s t f i v e days b e f o r e any expe r i m e n t a l procedure. T h i s p e r i o d o f time was s u f f i c i e n t f o r the animals to empty t h e i r guts. Two d r y i n g procedures were used. The f i r s t o f these was used f o r a l l animal m a t e r i a l s upon which i r o n a n a l y s es were t o be performed. I t c o n s i s t e d o f oven d r y i n g a t 110°C f o r 48.hours. The second procedure was used f o r a l l t i s s u e upon which chemical o r b i o c h e m i c a l a n a l y s e s were to be performed. T h i s procedure c o n s i s t e d o f s i x washes o f the t i s s u e i n each o f d i s t i l l e d water, 95% et h a n o l , acetone, and f i n a l l y e t h e r . A f t e r most o f the e t h e r fumes had evaporated, the t i s s u e was p l a c e d i n a vacuum d e s i c c a t o r over phosphorous p e n t o x i d e and sodium 5 hydroxide p e l l e t s for 48 hours. Wet weights of a l l tissues were determined p r i o r to drying procedures. Weight wets to 0.01 gm. were determined on a t r i p l e beam balance. Dry weights to 0.1 mgm. were determined on a Mettler Gram-atic Model B balance i n the presence of phosphorous pentoxide. Microscopic examination of tissue was done with a Zeiss GFL compound microscope under phase and brig h t f i e l d i l l u m i n a t i o n . Photomicrographs were taken through a Zeiss Photo-microscope on Adox KB 14 (A.S.A. 20). The following s t a t i s t i c a l methods were used where applicable; analysis of variance, Keul's sequential t e s t for mean differences, l i n e a r regression c a l c u l a t i o n and tests of the s i g n i f i c a n c e of calculated regression c o e f f i c i e n t s , 95% confidence l i m i t s i n calculated regressions, c o r r e l a t i o n c o e f f i c i e n t (r) and confidence l e v e l s i n the estimated (r) , Student's t - t e s t , t - t e s t for mean difference of paired observations (Snedecor, 1956; L i , 1964; Steel and Torrie, 1960). Since per cent data tends to be binomial in form, a l l such data, which were to be used for tests of significance, were transformed, and presented, as the angle corresponding to the arc sine square root of per cent (Li, 1964). F-ratios were considered s i g n i f i c a n t i f the p r o b a b i l i t y that the event was a random occurrence was equal to 0.05 or l e s s . P r o b a b i l i t y l e v e l s are presented with the data and non-significance i s denoted by the convention N.S. Other abbreviations used were S.D. for standard deviation and S.E. for standard error of the mean. A l l chemicals, other than biochemicals, were of reagent 6 grade. Para-dimethyl-amino-benzaldehyde,for Elson-Morgan reactions, was r e c r y s t a l l i z e d from reagent grade ethanol. The ethanol used for chemical tests was r e d i s t i l l e d . Hydrogen ion concentration (pH) was determined with a Beckman Research Model pH meter. Spectrophotometric and color-imetric data were determined on a Beckman DU 2 Spectrophotometer. Mechanical s l i t width was adjusted to supply a spectral s l i t width of 2 millimicrons at the wavelengths required for the p a r t i c u l a r determination. 2. H i s t o l o g i c a l Methods: For routine f i x a t i o n the buffered formalin with c e t y l pyridinium bromide i n d i s t i l l e d water (Culling, 1963) was used. Other f i x a t i v e s were 5% formalin i n sea water, Bouin's, Baker's, Carnoy's (Culling, 1963), and N-methyl morpholine with cyanuric chloride in methanol (Goland, _£t_al., 1967). Whole animals or tissues were fixed f o r 24 hours in a l l f i x a t i v e s except Carnoy's, i n which case maximum f i x a t i o n time was four hours. Whole animal preparations were s l i t occasionally along the longitudinal axis, or bisected across t h i s axis, to allow penetration of f i x a t i v e . Extremely large animals were injected simultaneously with f i x a t i v e upon immersion i n the f i x a t i v e . A f t e r f i x a t i o n tissues were dehydrated in graded ethanols, cleared i n benzene, and embedded i n Paraplast (M.P. 55.6°C). Trimmed blocks were cut routinely at 8 microns on a Spencer microtome. Frozen sections were cut from material which had been 7 fixed in c e t y l pyridinium-formalin for 12 hours at 10 or "16 microns on an International Cryostat. Although several hematoxylin stains were attempted, the chrome hematoxylin with phloxin method of Gomori (1941) was found to be most s a t i s f a c t o r y for routine h i s t o l o g i c a l examinations of tunic and whole animal sections. Mallory's t r i p l e s t a i n and Weigert's hematoxylin (Pantin, 1964) were used occasionally. Gomori's hematoxylin was used also for chromosome sta i n i n g (Melander and Wingstrand, 1953) . Blood smears were stained with Leishman's s t a i n . The smears were prepared by the following method. A small lon g i t u d i n a l i n c i s i o n was made in the subendostylar sinus area of the animal. A drop or two of blood was c o l l e c t e d from this i n c i s i o n and mixed with a drop of 5% formalin in sea water on a clean microscope s l i d e . The preparation was allowed to stand for a minute and then smeared across the s l i d e . Before the f l u i d dried, ten to twenty drops of Leishman's s t a i n in methanol were dropped onto the smear. The smear was allowed to stand for three minutes, then a volume of d i s t i l l e d water, equal to twice the volume of Leishman's stain, was dropped onto the s l i d e . A f t e r ten minutes the s l i d e was flooded with d i s t i l l e d water and very r a p i d l y dehydrated through two changes of 100% t e r t i a r y butanol, one change of 50% t e r t i a r y butanol 50% xylene, and cleared through two changes of xylene. After clearing, the smears were mounted immediately in Permount. Slides prepared i n t h i s manner disp l a y good s t a i n d i f f e r e n t i a t i o n , but the st a i n i n g q u a l i t y deteriorates af t e r several weeks. 8 Osmium f i x e d b l o o d c e l l s were prepared by exposing smears o f f r e s h b l o o d to osmium t e t r o x i d e vapours i n a s e a l e d c o p l i n j a r f o r one hour. A f t e r t h i s exposure, the smears were dehydrated, c l e a r e d , and mounted i n Permount f o r m i c r o s c o p i c examination under b r i g h t f i e l d i l l u m i n a t i o n . 3. H i s t o c h e m i c a l Methods: For the h i s t o c h e m i c a l i d e n t i f i c a t i o n o f i r o n , P e r l ' s method f o r f e r r i c i r o n , the T u r n b u l l B l u e method f o r f e r r i c i r o n , and the d i n i t r o r e s o r c i n o l method o f Humphrey (Pearse, 1961), were u t i l i z e d . Some s e c t i o n s to be s t a i n e d f o r i r o n were p r e t r e a t e d w i t h 30% hydrogen p e r o x i d e and 5% o x a l i c a c i d f o r two hours t o unmask p r o t e i n bound i r o n (Dales, 1965). For the d e t e c t i o n o f carbohydrates the f o l l o w i n g procedures were employed: a l c i a n b l u e w i t h n e u t r a l red, a l c i a n b l u e w i t h p e r i o d i c a c i d S c h i f f reagent (P.A.S.), P.A.S., Gomori's aldehyde f u c h s i n , ( C u l l i n g , 1963), t o l u i d i n e b l u e f o r metachromasia, methylene b l u e e x t i n c t i o n t e s t w i t h v e r o n a l a c e t a t e b u f f e r s from pH 1.63 - 9.05, Hale's d i a l y z e d i r o n method (Pearse, 1961), and azure A a t pH 1.5 and 4.0 (Szi r m a i , 1963). Some s e c t i o n s , t o be s t a i n e d w i t h a l c i a n b l u e , c o l l o i d a l i r o n , aldehyde f u c h s i n , t o l u i d i n e b l u e , and azure A, were a l s o methylated f o r 24 hours and s a p o n i f i e d ( C u l l i n g , 1963). P r o t e i n s were demonstrated h i s t o c h e m i c a l l y by the M i l l o n r e a c t i o n , the DDD SH method o f B a r n e t t and Seligman, and the mercury brom-phenol b l u e method o f Bonhag (Pearse, 1961). The presence o f h i s t o n e - l i k e p r o t e i n s , b a s i c p r o t e i n s , was 9 determined with Biebrich's s c a r l e t (Spicer, 1962). The methyl green and pyronin technique, with a ribonuclease control (Nutritional Biochemicals), was u t i l i z e d to demonstrate RNA (Pearse, 1961). Al c i a n blue with P.A.S. was used to s t a i n sections of tis s u e which had been submitted to either chemical or enzymatic degradation (see below) to determine i f such treatment unmasked either neutral or a c i d i c polysaccharides. 4. Hematological Methods: D i f f e r e n t i a l c e l l counts were made from Leishman stained smears of blood. The wet weight of the animal was recorded and two smears per animal were made. After staining, 100 c e l l s per s l i d e were counted for a t o t a l of 200 c e l l s per animal. A l l smears were counted under o i l immersion. The d i f f e r e n t i a l distributions of the blood c e l l types were counted for 80 animals over a wet weight range from < 1 gram to 80 grams. The mean per cent d i s t r i b u t i o n of each c e l l type i s given with the de s c r i p t i o n of that c e l l type based on the sample of 80 animals. Further, t h i s group of 80 animals was broken into four sub-groups? each sub-group consisted of 20 animals within a 20 gram wet weight range, i . e . 0-20 grams, 20-40 grams, 40-60 grams, 60-80 grams. An analysis of variance was performed for the d i s t r i b u t i o n of each c e l l type as a function of weight group. Per cent values for the c e l l d i s t r i b u t i o n were converted to the angle corresponding to the arc sine square root of per cent before these c a l c u l a t i o n s . I f a s i g n i f i c a n t F r a t i o was 10 obtained i n the analysis of variance, the means of per cent of the c e l l type, as the angle, were compared between weight groups for s i g n i f i c a n t differences by the sequential method of Keul. Hematocrits were estimated by the use of Kolmer centrifuge tubes. Hematocrits are expressed as volumes per cent of blood c e l l s packed to t o t a l volume of blood. C e l l s were packed by centrifuging a known volume of blood for 10 minutes at 3,000 r.p.m. on an Orbit Model 807 centrifuge. Total blood c e l l concentration per cubic millimeter of blood was estimated by the use of a hemocytometer. Duplicate counts were made for i n d i v i d u a l animals and the wet weight of the animal was recorded. Blood was taken d i r e c t l y into a white blood c e l l p i pette from an i n c i s i o n i n the subendostylar sinus area of the animal. The blood was d i l u t e d with 5% formalin i n sea water, to which had been added 0.1% eosin. The d i l u t i o n factor was 1:200. Plasma pH was determined on a Beckman Research Model pH meter upon plasma which had been separated from blood c e l l s by 10 minutes centrifugation at 3,000 r.p.m. on Orbit Model 807 centrifuge. 5. Tunic Structure and Injury Studies: Tunic was injured by making a shallow rectangular i n c i s i o n into the tunic of the body wall i n the r i g h t l a t e r a l area of the animal. The i n c i s i o n was approximately 5 mm. by 10 mm. with the long axis of the i n c i s i o n corresponding to the long axis of the animal. The s u p e r f i c i a l tunic, within t h i s area, was 11 l i f t e d from underlying tunic and epidermis. I f the epidermis was injured i n any way the sample was discarded. At 0, 1, 3, 5, 10, 15 and 20 days a f t e r the tunic injury, the Injured area with surrounding tunic and underlying tissues was excised from the animal and fixed i n c e t y l pyridinium bromide i n formalin. The excised tissue, a f t e r f i x a t i o n , was dehydrated, cleared, and embedded i n Paraplast. Sections were cut at 8 microns at r i g h t angles to the long axis of the o r i g i n a l injury. The sections were mounted and stained with chrome hematoxylin with phloxin counterstain (Gomori, 1941). Blood c e l l s i n the injured tunic were counted at the narrowest width between the external l i m i t s of the epidermis and the i n t e r n a l edge of the injury. This distance was measured with an ocular micrometer and a l l the c e l l s which were included within an o i l immersion f i e l d diameter (0.125 mm.) were counted over that distance. Six animals were used for each time period and the mean value of eight counts for each animal were used in the computation of c e l l numbers for that time period. When counting c e l l s on a given s l i d e , the counts were made on at l e a s t every fourth or f i f t h section i n order that error due to overlapping of c e l l s between adjacent sections be avoided. C e l l counts are expressed as mean number of c e l l s per cubic millimeter of tunic for the time period. Volume of tunic, in which the c e l l s were counted, was computed upon the basis of a known width of section, 8 microns, a known width of o i l immersion f i e l d , 0.125 mm., and the measured distance across the i n j u r y area. C e l l s , i n i n d i v i d u a l counts, were counted from the epidermis to the cut edge and tabulated as number of c e l l s per f i e l d . The f i r s t f i e l d would consist of a cube 0.125 mm. by 0.125 mm. by 8 microns in depth. The data could then be u t i l i z e d to express p o s i t i o n a l relationships within th injured area as well as c e l l density throughout the entire i n j u r y area. The data were also grouped into the inner versus outer h a l f of the injury area. This enabled the data to be treated as paired p o s i t i o n a l observations. The inner h a l f of the injured tunic i s the h a l f distance from the epidermis towards the exterior, the outer h a l f i s the remainder of the tunic i n the injured area. In addition to injured tunic, samples of normal body wall tunic were taken from the r i g h t l a t e r a l area of the animal to ascertain the d i s t r i b u t i o n of blood c e l l s and the structure of the uninjured tunic. Sections of these tissues were prepared and stained as with injured tunic. The width of tunic from epidermis to exterior of tunic was measured and c e l l s were counted by f i e l d from the epidermis to the periphery. Comparisons of c e l l concentration in blood and tunic were made from samples of animals i n a 20-30 gram range of wet weight. Nine r e p l i c a t e counts from each of six animals were used f o r the determination of mean c e l l concentrations i n the tunic. For the determination of c e l l p o s i t i o n in the tunic of the body wall, f i v e animals were selected which had tunic widths of approximately 0.625 mm. This width corresponds to f i v e f i e l d diameters under o i l immersion. Three counts were taken from widely separated sections of tunic of each animal, the mean concentration of c e l l s per f i e l d per animal was computed and used in c a l c u l a t i o n of the mean concentration from f i v e animals. For the studies of the number of laminae in the tunic as a function of wet weight, aldehyde fuchsin stained sections of tunic from 28 animals, ranging i n weight from 1.2 grams to 59.7 grams, were prepared.- The number of fibrous laminae from the epidermis to the l i m i t s of the tunic were counted i n s i x sections from each animal in the sample. The mean of these s i x counts was used i n s t a t i s t i c a l c a l c u l a t i o n s . The author uses the term 'stolon' to ref e r to tube-like projections of tunic, epidermis, and sub-epidermal blood spaces which grow from the base of the animal to form a hold-fast. Types and numbers of blood c e l l s in the tunic of the stolon were determined i n the same manner as they were i n injured tunic and normal tunic of body w a l l . The stolon was l i g a t e d at i t s base, adjacent to the body of the animal, and then excised proximal to the l i g a t i o n . The stolon was fixed, dehydrated, embedded, and stained with chrome hemotoxylin. Stolons, u t i l i z e d for c e l l p o s i t i o n and concentration data, do not represent 'natural' h o l d - f a s t material, but are stolons which grew from the base of the ascidian i n the laboratory. The counts of c e l l s , and t h e i r r e l a t i v e p o s i t i o n i n the tunic of the stolon, were determined from sections taken approximately at the h a l f distance between the body wall and d i s t a l end of the stolon. 14 6 j Garbon Phagocytosis: A Suspension of Aqua-dag c o l l o i d a l carbon, 5% s o l i d s i n f i l t e r e d sea water, was injected into the tunic material of the l e f t l a t e r a l area of the tunicates. The carbon suspension was injected into the tunic proper; i f , as determined by subsequent sectioning of the tunic, the carbon was found to be injected b§low the epidermis, the sample was discarded; At 0, 3, 6, 12, 18j 24 and 48 hours aft e r carbon i n j e c t i o n , blood was taken from the subehdostylar s inus, and fresh and Leishman stained smears were made. The type of blood c e l l s which showed the presence of carbon p a r t i c l e s were determined from the Leishman Smears and by the examination of fresh smears under bri g h t f i e l d and phase i l l u m i n a t i o n . A t each of the time periods, 6 animals w'ejfe bled and two smears of fresh blood per ahimal were prepared i Twenty o i l immersion f i e l d s , ten per s l i d e , were @§uhted f o r each animal. The number of. the p a r t i c u l a r c e l l types with carbon p a r t i c l e s were determined and expressed as the per cent of that c e l l type with carbon p a r t i c l e s . Per @efit data were transformed by the arc sine method before any @©mputat-iohs were done. 7« Chemical Analyses: Tunicih refers to the carbohydrate material derived from a§@idiari tunic through the action of acid and base. Tunicin was prepared from the tunic of Halocyntbia aurantium in the following manner. Fresh tunic, excluding the tunic of the §i-ph©hs and hold-fast/ was stripped from the ascidian, b l o t t e d 1 5 dry, wet weighed, washed with d i s t i l l e d water, ethanol, acetone, and ether, and dried i n vacuum over phosphorous pentoxide and sodium hydroxide. Dry tunic was placed i n IN HCl for 4 8 hours in the r e f r i g e r a t o r . The mixture was s t i r r e d occasionally during t h i s period. The tunic was removed from the acid and washed with d i s t i l l e d water u n t i l the wash was neutral to pH paper. Tunic material was placed i n IN NaOH for 36 hours at 1 0 0 ° C . Every 1 2 hours during t h i s period, the base was decanted, and fresh base added. After 36 hours, the base was decanted and the residual t u n i c i n was washed with d i s t i l l e d water u n t i l neutral to pH paper. The t u n i c i n was washed with ethanol, acetone, and ether. After the ether had evaporated, the t u n i c i n was placed in a vacuum desiccator and dried for 4 8 hours over phosphorous pentoxide and sodium hydroxide. This preparation of tunic i n , p a r a l l e l s the preparation of c h i t i n from lobster s h e l l (Hackman, 1 9 5 4 ) . A f ter 4 8 hours, y i e l d of t u n i c i n was determined by dry weighing the product. Material prepared i n t h i s manner was u t i l i z e d for chemical and biochemical analyses. Tissues to be analyzed for iron were oven dried for 4 8 hours. A l l iron values are expressed as micrograms of iron per gram dry weight or parts per m i l l i o n dry weight. After drying, the material was weighed and wet ashed with s u l f u r i c and n i t r i c acids on a Kjeldahl burner. Pe r c h l o r i c acid was added to the digest mixture a f t e r preliminary digestion of the tissue was complete. The acid digestion solution was heated u n t i l almost dry, at which point i t was cooled, 16 d i l u t e d with d i s t i l l e d water, and neutralized with sodium hydroxide in an ice bath. Aliquots of t h i s neutralized mixture were used for the iron determinations. The iron was determined by the co l o r i m e t r i c o-phenanthroline method (Sandell, E.B., 1950). The d i s t r i b u t i o n of iron i n the various tissues was determined by d i s s e c t i n g these tissues from the animal, drying them, and determining the iron as above. The tissues of several animals generally were pooled for these iron estimations. Iron concentration of the blood c e l l s was determined by pooling the c e l l s from several animals. For d i a l y s i s studies, c o l l e c t e d blood c e l l s were submitted to sonic v i b r a t i o n and homogenation i n a known volume of d i s t i l l e d water. After t h i s procedure, the homogenate was spun for 10 minutes at 3,000 r.p.m;. on an Orbit Model 807 centrifuge. The super-natant was dialyzed for 48 hours against d i s t i l l e d water in the r e f r i g e r a t o r . The dialyzate, a f t e r oven drying, and the d i s t i l l e d water were analyzed for ir o n . The d i s t i l l e d water was concentrated by b o i l i n g before the iron analysis. The spectrographic analysis of pooled blood c e l l s for iron, vanadium, titanium, chromium, columbium and manganese was done by Coast Eldridge Engineers, Vancouver, B r i t i s h Columbia. Dried tunic and t u n i c i n were analyzed as well for per cent composition of carbon, hydrogen, oxygen, nitrogen and s u l f u r . Tunicin was hydrolyzed in 72% s u l f u r i c acid (v/v) at room temperature (Saeman, et a l . , 1963). Aliquots of the hydrolyzate 17 were taken at 0.5, 1, 3, 5, 7, 9' and 11 hours and neutralized with sodium hydroxide in an ice bath. Reducing sugars i n hydrolysates were estimated by a modified Somogyi method (Hestrin, 1963). Aliquots from 0.5, 3, and 9 hours were analyzed for the presence of uronic acids according to the carbazole method of B i t t e r and Muir (1962). The sugars present in the hydrolysates were i d e n t i f i e d , a f t e r n e u t r a l i z a t i o n with base, by ascending t h i n layer chromatography (Ovodov, 1967). S i l i c a gel G plates were spread at 0.25 mm., and 10 to 20 m i c r o l i t e r s of the neutralized hydrolysate and known sugars or sugar acids (Nutritional Biochemicals) were applied to the pla t e s . Two solvent systems were employed: the f i r s t system, for uronic acids, consisted of n-butanol: ethanol: 0.1N HCl (1:10:5 v/v); the second system, for reducing sugars consisted of n-butanol: acetone: water (4:5:1 v/v) (Ovodov, 1967). The plates were developed with a n i l i n e hydrogen phthalate or s u l f u r i c acid (Stahl, 1965) . Hexosamines are acid as well as base l a b i l e . The hy d r o l y t i c y i e l d of hexosamines during acid hydrolysis i s a function not only of the l i b e r a t i o n of the hexosamine from the substrate, but also the degradation of hexosamine to furan d e r i v a t i v e s . As a res u l t , hydrolysis procedures for the estimation of the hexosamine content of tissue necessitate that a preliminary study of the optimal time of hydrolysis for maximum y i e l d be determined (Neuberger and Marshall, 1966). Time curves of y i e l d were prepared for the hydrolysis of dried tunic in 6N HCl at 2, 4, 6 and 8 hours at 100°C. T r i p l i c a t e samples of tunic were sealed in pyrex te s t tubes with 1 ml. of acid and hydrolyzed for each time period. Acid hydrolysis of tissue also w i l l release peptides and amino acids which i n t e r f e r e with the Elson-Morgan reaction for hexosamines. Therefore, af t e r tunic hydrolysis, the hydrolysate was decontaminated by passing i t through a column of Dowex 50W X-8 cation exchange r e s i n i n the hydrogen form (Boas, 1953). The eluates of these columns were analyzed by a modified Elson-Morgan reaction (Boas, 1953) for hexosamines. Hexosamine mixtures can be separated into t h e i r constituent amino sugars by controlled e l u t i o n through a long column, 48 cm. by 0.8 cm., of Dowex 50W X-8 with 0.30N or 0.33N HCl and the c o l l e c t i o n of s e r i a l f r a c t i o n s of the eluate (Gardell, 1961; Crumpton, 1959). From t h e i r r e l a t i v e e l u t i o n volumes, the concentration of 2-amino-D-glucose and 2-amino~D-galactose i n the hydrolysates was determined. Spectral absorption curves were determined for a l l samples submitted to the Elson-Morgan reaction to v e r i f y that the colored product was hexosamine (Boas, 1953). Standards were prepared and treated, excluding hydrolysis, i n the same manner as unknowns. 2-amino-D-glucose and 2-amino-D-galactose were obtained from C a l i f o r n i a Biochemicals. Tunic, before and a f t e r pronase digestion (see below), was hydrolyzed and the amino acid composition determined. Approximately f i v e milligrams of dried tunic were sealed in a pyrex t e s t tube under vacuum, 0.01 mm. Hg., with 1 m i l l i l i t e r of 6N HCl and hydrolyzed for 24 hours at 105° + 1°C. After 19 the hydrolysis, the t e s t tube was opened and the hydrolysiate was aspirated o f f the remaining tunic material. The residue was washed once with d i s t i l l e d water and the wash was combined with the o r i g i n a l hydrolysate. The hydrolysate was deproteinized with s u l f o s a l i c y l i c acid, m i l l i p o r e f i l t e r e d , pore diameter 0.4 microns, and centrifuged at 40,000 times gravity in a Serval centrifuge. The supernatant was aspirated o f f and evaporated to dryness i n a vacuum over sodium hydroxide and concentrated s u l f u r i c acid. The dried hydrolyzate was taken up i n 0.5 ml. of 0.01N HCl. 0.2 ml. of t h i s solution was used for the chromotography of a c i d i c and neutral amino acids, and 0.2 ml. were used for the separation of the basic amino acids. The amino acids were separated and i d e n t i f i e d by chromotography on a Spinco Amino Acid Analyzer, Model C. A c i d i c and neutral amino acids were chromatographed on a 54.9 cm. column of Spinco UR-30 r e s i n . The system uses two buffers and two temperatures which are programmed to elute the amino acids over temperature and pH gradients. The buffers used for the e l u t i o n of a c i d i c and neutral amino acids are 0.20 N sodium c i t r a t e at pH 3.158 and pH 4.208. The change in buffers takes place at 170 minutes a f t e r the i n i t i a t i o n of a 360 minute e l u t i o n . The e l u t i o n rate was 50 ml./hour for the b u f f e r s . A temperature change from 32.5°C to 62.5°C was programmed for 87 minutes a f t e r the s t a r t of the e l u t i o n . The basic amino acids were eluted on a 21.8 cm. column of Spinco PA-35 r e s i n . The buffers used in the basic amino acid e l u t i o n are 0.38N sodium c i t r a t e , pH 4.246, and 0.35 N sodium c i t r a t e , pH 5.249. 20 The programmed buffe r change i s at 250 minutes of 360 minutes t o t a l e l u t i o n time. A temperature change from 32.5°C to 55.0°C started at 130 minutes a f t e r the i n i t i a t i o n of e l u t i o n . The flow rate was 50 ml./hour for the buffe r s . Upon elut i o n of the amino acids from the columns, they are subjected to the ninhydrin reaction, ninnydrin flow rate 25 ml./hour, and the o p t i c a l density of the reaction product i s recorded and integrated at 570 and 440 millimicrons wavelength. Standards, co n s i s t i n g of known amino acids, were eluted on the amino acid analyzer to ascertain t h e i r r e l a t i v e e l u t i o n times and to c a l i b r a t e integration output with known amino acid concentration. The amino acids, for the standards, were obtained from C a l i f o r n i a Biochemicals. The amino acid composition of tunic, before and a f t e r pronase digestion, was presented as the molar per cent of t o t a l amino acids i n the hydrolysates, and as per cent of amino acids present which are pronase susceptible or l a b i l e i n the tunic. Dried tunic was treated for 7 days with 4% sodium hydroxide at room temperature, a f t e r which i t was washed with d i s t i l l e d water, 0.5% a c e t i c acid, and d i s t i l l e d water u n t i l the washings were neutral. After t h i s procedure, the prepared tunic was acetylated with acetic anhydride, g l a c i a l acetic acid, and s u l f u r i c acid (1:1:1.05 v/v) for twenty minutes on a b o i l i n g water bath (Hestrin, 1963). Pigment was extracted from tunic, which had been washed with d i s t i l l e d water, by immersing the tunic in methanol under a nitrogen atmosphere. After several such extractions, the 21 methanol, with the pigment, was extracted further with n-hexane under nitrogen. The absorption spectra of the methanol and hexane extracts were measured at 5 mi l l i m i c r o n wavelength increments from 300 to 800 millimicrons on a Beckman DU 2 Spectrophotometer. Pooled blood c e l l s were submitted to spectral examination also. Pooled blood c e l l s were extracted with methanol under nitrogen and the extracts were centrifuged in sealed tubes for 10 minutes at 3,000 r.p.m. on an Orbit Model 807 centrifuge. The supernatant methanol, containing the pigment, was extracted further with n-hexane under nitrogen. The methanol and hexane extracts were examined spectrophoto-m e t r i c a l l y under the same wavelengths as tunic extracts. Tunicin was treated with hot, concentrated l i t h i u m thiocyanate (Got±schalk,1966) and Schweitzer's reagent at room temperature for 48 hours (Jayme and Lang, 1963). Tunicin, and pronase treated tunic, were treated with 85% H^PO^ for 8 hours at 10°C (Jayme and Lang, 1963). 8. Enzymatic Analyses: Pronase, c e l l u l a s e (Aspergillus niger), chitinase (Actinomyces), hyaluronidase (bovine testes) , papain, and tr y p s i n were obtained from C a l i f o r n i a Biochemicals. Hyaluronic acid was obtained from N u t r i t i o n a l Biochemicals. A l l enzyme assays or digests of tunic material or t u n i c i n were performed at l e a s t in t r i p l i c a t e . The blanks for product determinations of tunic or t u n i c i n digestions were incubated enzyme blanks which contained a l l the ingredients of the tunic digestion system except substrate. 22 The papain and t r y p s i n treatment of tunic consisted of two steps. The f i r s t of these involved a pre-incubation of the tunic i n 0.1M acetate buffer, pH 5.5, which was 0.005 M i n cysteine-HCl and 0.005 M in sodium versenate, at 60°C for 30 minutes. At the end of t h i s period, 2 milligrams of papain per gram of tunic were added to t h i s medium and i t was incubated in a constant temperature water bath for 24 hours ( S c h i l l e r , et a l . , 1961). Tunic material was c o l l e c t e d from the papain digest and washed repeatedly with d i s t i l l e d water. The tunic was digested with trypsin, 2.5 milligram of enzyme per gram of tissue, i n 0.1 M phosphate buffer for 72 hours ( S c h i l l e r , et a l . , 1954). After the 72 hour incubation period, the tunic was removed from the incubation medium, washed repeatedly with d i s t i l l e d water to free i t of buffer and enzyme, dehydrated i n graded alcohols, cleared, embedded, and sectioned for h i s t o l o g i c a l and histochemical examination. Tissues prepared i n t h i s manner were stained with a combination A l c i a n Blue-P.A.S. s t a i n (Culling, 1963). Tunic was digested with pronase, 0.5 milligram of enzyme per milligram of tissue, i n 0.15 M phosphate buffer at pH 8.0 which was 0.005 M i n calcium chloride, for 48 hours at 37°C. The incubation medium, minus the tunic, was pre-incubated for one hour before the addition of tunic (Baker and Young, 1966; Inoue and Yosizawa, 1966). Dry weight of tunic, before and a f t e r pronase digestion, was ascertained. Tunic, a f t e r pronase digestions, was submitted to c e l l u l a s e , hyaluronidase, or chitinase digestion. Pronase digested tunic also was examined histochemically by the A l c i a n Blue-P.A.S. technique (Culling, 1963) . Tunic, pronase digested tunic, and t u n i c i n were treated with c e l l u l a s e . The incubation medium for c e l l u l a s e digestion consisted of 0.1 M acetate buffer at pH 5.0. The enzyme to substrate r a t i o was approximately 1 milligram of enzyme per 10 milligrams of tunic or t u n i c i n . The tissue was incubated with the enzyme at 50°C for the appropriate time period in a constant temperature water bath in sealed tubes (H a l l i w e l l , 1961; Reese and Mandels, 1963). Pronase digested tunic and t u n i c i n were submitted to a preliminary c e l l u l a s e digestion for 28 hours. At the end of t h i s period, the remaining tunic material was c o l l e c t e d by centrifugation. The residue was washed repeatedly with d i s t i l l e d water and placed in 85% H^PO^ at 10°C for 8 hours. The phosphoric acid mixture was centrifuged, the acid was aspirated, and the residue was washed repeatedly with d i s t i l l e d water u n t i l the wash was neutral to pH paper. The pronase treated tunic or t u n i c i n were again submitted to c e l l u l a s e digestion for 48 hours, a f t e r which the residue was separated from the incubation medium and treated again with phosphoric acid and submitted to c e l l u l a s e d i gestion for a further 24 hours. At the end of the 24 hours, t h i s procedure was repeated, and the tunic residue was submitted to a f i n a l 48 hours of c e l l u l a s e digestion. The t o t a l c e l l u l a s e incubation time for these samples was 148 hours. Aliquots of the digestion mixtures were taken at 4, 8, 12, 20, and 28 hours of the preliminary incubation, at 12, 24, 36, and 48 hours of the second digestion, and at the end of the t h i r d 24 (24 hours) and f i n a l (48 hours) digestion periods. Further samples of t u n i c i n were treated with phosphoric acid and submitted to 14 days incubation with c e l l u l a s e . At the end of t h i s period of time, the supernatant was assayed for reducing sugars, as were the e a r l i e r digests, by a modified Somogyi method (Hestrin, 1963). The supernatant of the 14 day c e l l u l a s e incubation was deproteinised by b o i l i n g , and centrifuged. The supernatant was chromatographed on thi n layer S i l i c a Gel plates to i d e n t i f y the constituent reducing sugars (Ovodov, 1967). Tunicin, t u n i c i n which had been treated with phosphoric acid, tunic, and pronase treated tunic were submitted to hyaluronidase digestion. The enzyme to substrate r a t i o i n t h i s procedure was approximately 1:10. The incubation medium was 0.2 M phosphate buffer at pH 6.8, and the incubation temperature was 37°C (Dorfman, 1955). The solutions were digested for 14 days i n sealed tubes i n a constant temperature bath. After the fourteen days, the supernatant was analyzed for reducing sugars (Hestrin, 1963) and hyaluronate by a modified Morgan-Elson reaction (Greiling, 1965). The tunic and t u n i c i n materials remaining a f t e r the digestion period were washed thoroughly with d i s t i l l e d water, dehydrated through graded alcohols, cleared, embedded, sectioned, and stained with A l c i a n Blue-P.A.S. for histochemical examination. Pronase treated tunic and t u n i c i n were submitted to chitinase digestion for fourteen days at 37°C in 0.1 M acetate buffer at pH 5.0 (Tracey, 1955). The enzyme to substrate 25 r a t i o was approximately 1:10. Over the incubation period, aliquots of the supernatant were assayed for reducing sugars (Hestrin, 1963) and hexosamine (Boas, 1953). 26 RESULTS 1. Wet versus dry weight: There i s a l i n e a r r e l a t i o n s h i p between the wet and dry weights of the tunicate Halocynthia aurantium. Based on a sample of 29 animals, the calculated c o r r e l a t i o n c o e f f i c i e n t (r) i s + 0.997. Regression analysis of the data reveals a slope value of 0.0649. The p r o b a b i l i t y that the calculated slope value does not represent the population slope value i s less than 0.001. 2. The Cytology of the Blood: Ten blood c e l l types are recognized in blood of H. aurantium based on phase contrast and b r i g h t f i e l d microscopy of l i v e c e l l s and Leishman stained smears of blood c e l l s . (a) Mature Morula C e l l (M.M.): The mature morula c e l l s comprise 2.9% of the blood c e l l s i n c i r c u l a t i o n based on a sample of 80 animals. These c e l l s are between 9 and 10 microns i n diameter. The nucleus i s never observed to have a nucleolus, although the nucleus i s often obscured. The mature morula c e l l i s f i l l e d with vacuoles or v e s i c l e s approximately 1 to 2 microns i n diameter. The number of these inclusions varies, but i s seldom less than twenty. Under brig h t f i e l d i l l umination the mature morula c e l l is highly r e f r a c t i v e and appears to be l i g h t green in color. Phase contrast studies reveal even greater r e f r a c t i v i t y i n the mature morula. The content of the vacuoles i s homogenous under b r i g h t f i e l d and phase contrast, and i s highly a c i d o p h i l i c with Leishman's s t a i n . Osmium fixed smears of mature morula c e l l s display a l i g h t brown to gray 27 color a t i o n of the vacuoles. The mature morula i s distinguished from other vacuolar c e l l s by the great number of i t s inclusions, t h e i r high r e f r a c t i v i t y , t h e i r a c i d o p h i l i c nature, and the small s i z e of the vacuoles, i . e . always smaller than the nucleus when i t i s v i s i b l e (Fig. 1). (b) Immature Morula C e l l s (I.M.): The immature morula c e l l s , which are approximately 9 microns in diameter, comprise 28.1% of the blood c e l l s in c i r c u l a t i o n . This c e l l i s characterized by the fact that i t i s f i l l e d with 5 to 15 vacuoles which are on the same order of size, 3 microns, as the nucleus. The s t a i n i n g reactions of the vacuoles are s i m i l a r to the mature morula vacuoles; they are highly a c i d o p h i l i c and turn grey aft e r exposure to osmium vapors. The vacuoles are a homogeneous l i g h t green under bri g h t f i e l d i l l u m i n a t i o n and di s p l a y some r e f r a c t i v i t y , but i t i s less than i n mature morula c e l l s . C e l l s , which appear to be t r a n s i t i o n a l i n number and size of vacuoles, between the mature and immature morula, are evident i n blood smears and were counted as immature morula c e l l s i f the vacuoles present were equal to or larger than the nucleus in diameter and were f i v e or more i n number (Fig. 1) . (c) Compartment C e l l s (Comp.): The compartment c e l l , 1.0% of the blood c e l l s , i s about 8 microns i n diameter and has 2 to 4 large vacuoles a l l of which are at l e a s t 3 microns i n diameter and larger than the nucleus. The vacuoles, which f i l l the c e l l , are strongly a c i d o p h i l i c and appear l i g h t green i n l i v e smears. These vacuoles are also s l i g h t l y r e f r a c t i v e and 28 homogeneous i n content with no apparent granular inclusions. The nucleus does not display a nucleolus. These c e l l s are distinguished from immature morula c e l l s based on the larger s i z e of the vacuoles and the low number of them (Fig. 1) . (d) Signet Ring C e l l s (signet): The signet r i n g has one large vacuole which f i l l s the cytoplasm, thus squeezing the nucleus to one side of the c e l l . These c e l l s are on the order of 6 microns i n diameter and comprise 1.7% of the blood c e l l s . A c e l l i s considered to be a signet type when the vacuole i s larger than the nucleus and when the vacuole does not contain any granular inclusions. The vacuole may s t a i n either acidophile or neutral. The nucleus has been seen occasionally to contain a small nucleolus (Fig. 1). (e) Hyaline Amoebocytes (H.A.): The hyaline amoebocytes comprise 38.5% of the blood c e l l s and range from 6 microns to 12 microns i n diameter. The nucleus i s often nucleolated. The cytoplasm i s homogeneously neut r o p h i l i c with Leishman's stain and i t may contain one or more vacuoles which do not s t a i n . Under b r i g h t f i e l d illumination, the cytoplasm of l i v e c e l l s ranges from hyaline to f i n e l y granular. The vacuoles i n t h i s case, as well as i n phase contrast studies, may contain one or two small granular concretions which are subject to Brownian motion. Under phase illumination, the cytoplasm appears r e t i c u l a t e d and even appears clumped i n some cases. Within the hyaline amoebocytes, large yellow granules may often be observed. These c e l l s are very amoebocytic and assume a v a r i e t y of b i z a r r e shapes when viewed in l i v e smears (Fig. 1). 29 (f) Granular Amoebocyte (G.A.): The granular amoebocytes make up 20.5% of the c e l l s in c i r c u l a t i o n . They have a dis c r e t e s i z e range, 6-8 microns, and are packed with d i s t i n c t granules, 0.2 microns i n diameter, which have some r e f r a c t i v e properties when viewed under bright f i e l d i l l u m i n a t i o n . In Leishman stained smears the granules s t a i n an intense neutrophile and are seen not to f i l l the cytoplasm, but leave a peripheral hyaline layer. This peripheral hyaline layer i s further v e r i f i e d i n phase contrast studies of l i v e c e l l s . Under phase illumination the granulation i s seen to be associated with a r e t i c u l a r network. The nucleus often displays a small nucleolus (Fig. 1). (g) Stem C e l l (Stem): The stem c e l l i s the smallest c e l l , 5-6 microns, observed i n c i r c u l a t i o n . This c e l l makes up 4.8% of the blood c e l l s . The nucleus, which almost f i l l s the c e l l , may contain one or two small n u c l e o l i . The chromatin i s disperse and the cytoplasm stains a homogeneous dense ba s o p h i l i c with Leishman's s t a i n . Under b r i g h t f i e l d and phase illumination, the.cytoplasm of l i v e c e l l s appears hyaline and the nucleus looks clear except for the n u c l e o l i . (h) Stem C e l l with A c i d o p h i l i c Vacuoles or Granules (S.A.V.G.): This c e l l i s e s s e n t i a l l y s i m i l a r to the stem c e l l except that i t reaches a s l i g h t l y larger size, 5-7 microns, and possesses either granules or vacuoles which are acidophile to Leishman's s t a i n . The vacuoles, i f present, are always smaller than the nucleus which may have one or two n u c l e o l i . The granules appear to be a dense black color when viewed under phase contrast i n l i v e c e l l s . This c e l l type makes up 1.0% of the blood c e l l s . The stem c e l l s with a c i d o p h i l i c vacuoles or granules have been observed to have both the vacuoles and granules (Fig. 1). (i) Giant Stem C e l l : This c e l l very r a r e l y i s observed in blood smears. I t comprises 0.2% of the blood c e l l s and i s quite large, 9-10 microns in diameter. I t has a large nucleus, 5 microns i n diameter, which may have a large nucleolus. The stai n i n g reaction of the giant stem type are e s s e n t i a l l y the same as the stem c e l l type. Under b r i g h t f i e l d illumination, l i v e giant stem c e l l s are quite amoeboid and the cytoplasm i s hyaline to f i n e l y granular. (j) Dispersed Vesicular C e l l (D.V.C.): The dispersed v e s i c u l a r c e l l appears to be of a uniform diameter, 8 microns, and comprises 1.7% of the blood c e l l s . The cytoplasm contains generally 8-12 v e s i c l e s of s i m i l a r size, 1 micron i n diameter, which are regul a r l y dispersed throughout the c e l l . These v e s i c l e s are d i f f i c u l t to observe in l i v e c e l l s under bright f i e l d illumination, but under phase contrast they appear as l i g h t homogeneous v e s i c l e s against a dark cytoplasmic back-ground. With Leishman 1s s t a i n , the cytoplasm displays a l i g h t b a s o p h i l i a and the v e s i c l e s , a l i g h t e o s i n o p h i l i a . With Gomori's hemotoxylin-phloxin stain, the v e s i c l e s s t a i n an intense blue black. I f the oxidation step in t h i s procedure i s omitted, the v e s i c l e s take an intense phloxin s t a i n . The nucleus w i l l occasionally have a nucleolus (Fig. 1). 3. Hematology: The mean number of c e l l s per cubic millimeter of blood 31 FIGURE 1. The blood c e l l s of H. aurantium. MM, mature morula c e l l ; IM, immature morula c e l l ; GA, granular amoebocyte; S i , signet r i n g c e l l ; HA, hyaline amoebocyte; C, compartment c e l l ; SAVG, stem c e l l with a c i d o p h i l i c vacuoles or granules; DV, dispersed v e s i c u l a r c e l l . A. Live c e l l s , phase contrast. 3,500X. B. Live c e l l s , phase contrast. 2,500X. C. Leishman's stained blood smear, bright f i e l d . 2,500X D. Live c e l l s , phase contrast. Pressure has been applied to the cover s l i p . Note the nucleolus. 2,500X. E. Live c e l l s , phase contrast. Note the nucleolus and the homogeneous nature of the v e s i c l e s . 2,500X. F. Live c e l l s , phase contrast. Pressure has been applied to the cover s l i p . Note the anucleolar n u c l e i i n the MM c e l l s . 1,500X. from H. aurantium, as determined by duplicate hemocytometer counts of the blood from 35 animals, i s 17,240 with a standard error of 1,690. The analysis of variance of number of c e l l s per cubic millimeter of blood between twenty gram wet weight groups from 0-80 grams demonstrates an i n s i g n i f i c a n t F r a t i o . The hematocrit mean, or mean per cent of blood volume as c e l l s , i s 0.38% (n = 15) with a standard error of 0.06%. The mean corpuscular volume calculated from hematocrit and c e l l concentration data i s 220 cubic microns. Mean plasma pH (n = 6) i s 7.31 with a range of 7.20 - 7.41. The d i f f e r e n t i a l d i s t r i b u t i o n of blood c e l l types, derived from the 80 animal sample, i s given with the description of these c e l l s (see above). The analysis of variance of per cent d i s t r i b u t i o n of p a r t i c u l a r c e l l types as a function of 20 gram wet weight groups displays s i g n i f i c a n t F r a t i o s i n the following categories of c e l l s : mature morula (F r a t i o = 6.188, P < 0.005), stem c e l l (F = 3.752, P < 0.025), stem with a c i d o p h i l i c vacuoles or granules (F = 7.173, P <i 0.005), dispersed v e s i c u l a r c e l l (F = 3.969, P < 0.025), and hyaline amoebocyte (F = 13.286, P < 0.005). The compartment c e l l , the immature morula, and signet r i n g c e l l s do not show s i g n i f i c a n t F r a t i o s between weight groups when considered as i n d i v i d u a l categories. But, i f these c e l l s are grouped together into a single category of 'vacuolar c e l l s ' , the analysis of variance reveals a s i g n i f i c a n t F r a t i o i n d i s t r i b u t i o n as a function of weight group (F = 2.789, P < 0.05). The r e l a t i v e numbers of stem c e l l , stem with a c i d o p h i l i c vacuoles or granules, vacuolar c e l l s , mature morula and dispersed v e s i c u l a r c e l l decrease with increased weight (Fig. 2). The hyaline amoebocytes increase with increased FIGURE 2 . The per cent d i s t r i b u t i o n of blood c e l l types i n blood as a function of 2 0 gram whole animal wet weight groups. Per cent data have been transformed to the angle corresponding to the arc sine square root of per cent. Each point is the mean value for twenty animals in that weight group. Vacuolar c e l l s refer to a c o l l e c t i v e group including the immature morula, the compart-ment, and the signet r i n g c e l l . o1 0-20 20-40 40-60 60-80 WEIGHT GROUP IN G R A M S 34 weight of animal (Fig. 3). The means of the 20 gram wet weight groups for each c e l l type which displayed s i g n i f i c a n t F r a t i o s were compared for s i g n i f i c a n t differences by the sequential method of Keul. These mean comparisons are presented i n Tables 1 through 6, with the p r o b a b i l i t y l e v e l s of s i g n i f i c a n t differences. The per cent means of p a r t i c u l a r c e l l types i n each 20 gram wet weight group are given i n Table 7. 4. Morphology of the Tunic: The tunic of H. aurantium i s considered to consist of three components: the epidermis, the tunic matrix, and the c u t i c u l a r spinous processes born externally on the tunic surface (Fig. 4). Further, the tunic can be considered as tunic of the body wall, t u n i c B , and tunic of the stolon, tunicg, (see below). The thickness of the tunic shows great v a r i a t i o n . A range of values have been observed from 0.60 mm. thickness from a 1.5 gram animal (wet weight) to 1.20 mm. for a 60 gram animal. (a) The Epidermis: The epidermis of the body wall i s columnar, but i s highly variable i n form and appears to be cuboidal i n the stolons. The cytoplasm i s basophilic when stained with chrome hemotoxylin and phloxin. The nuclear chromatin i s d i f f u s e and a nucleolus i s observed r a r e l y . The c e l l s measure approximately 8 by 6 microns. Frontal sections of the epidermis reveal that the c e l l s are polygonal in outline and are surrounded by at l e a s t 6 other c e l l s . No pores, channels, or vessels penetrate the epidermis. Fibers are seen to protrude from the apical ends of the epidermal c e l l s into 35 FIGURE 3. The per cent d i s t r i b u t i o n of hyaline amoebocytes in blood as a function of 20 gram whole animal wet weight group. Per cent data has been transformed by the arc sine method. Each point i s the mean value determined for 20 animals i n that weight group. 36 TABLE 1. The s i g n i f i c a n c e of differences (Keul's method) of mean per cent of stem c e l l s i n blood between 20 gram wet weight groups of H. aurantium. A l l per cent data has been transformed by the arc sine method. Significance l e v e l s are noted below mean differences in the table. X Angle X-4 X-3 X-2 WEIGHT GROUP No. Range 1 0-20 grams 14.25 5.30 3.61 2.25 .01 .01 .01 2 20-40 grams 12.01 3.06 1.37 .01 .01 3 40-60 grams 10.64 1.69 .01 4 60-80 grams 8.95 37 TABLE 2. The s i g n i f i c a n c e of differences (Keul 1s method) of mean per cent of stem c e l l s with a c i d o p h i l i c vacuoles and granules in blood between 20 gram wet weight groups of H. aurantium. A l l per cent data have been transformed by arc sine method. Significance l e v e l s are noted below mean differences in the table. WEIGHT GROUP No. Range X Angle X-4 X-3 X - 2 1 0-20 grams 7.25 4.37 2.83 2.75 .01 .01 .01 2 20-40 grams 4.50 1.62 0.08 .01 N.S. 3 40-60 grams 4.42 1.54 .01 4 60-80 grams 2.88 38 TABLE 3. The s i g n i f i c a n c e of differences (Keul's method) of mean per cent of 'vacuolar' c e l l s in blood between 20 gram wet weight groups of H. aurantium. Per cent data have been transformed by the arc sine method, Significance l e v e l s are noted below mean differences in the table. X Angle X-4 X-3 X-2 WEIGHT GROUP No. Range 1 0-20 grams 36.28 5.36 5.13 2.42 .01 .01 .01 2 20-40 grams 33.86 2.94 2.71 .01 .01 3 40-60 grams 31.15 0.23 N.S. 4 60-80 grams 30.92 39 TABLE 4. The s i g n i f i c a n c e of differences (Keul's method) of mean per cent of mature morula c e l l s in blood between 20 gram wet weight groups of H. aurantium. Per cent data has been transformed by the arc sine method. Significance l e v e l s are noted below mean differences in the table. X Angle X-4 X-3 X-2 WEIGHT GROUP No. Range 1 0-20 grams 12.02 5.44 4.62 3.91 .01 .01 .01 2 20-40 grams 8.11 1.53 0.71 .01 .05 3 40-60 grams 7.40 0.82 .01 4 60-80 grams 6.58 40 TABLE 5. The s i g n i f i c a n c e of differences (Keul's method) of mean per cent of dispersed v e s i c u l a r c e l l s i n blood between 20 gram wet weight groups of H. aurantium. Per cent data has been transformed by the arc sine method. Significance l e v e l s are ' noted below mean differences in the table. X Angle X-4 X-3 X-2 WEIGHT GROUP No. Range 1 0-^ -20 grams 8.24 3.36 3.02 1.19 .01 .01 .01 2 20-40 grams 7.05 2.17 1.83 .01 .01 3 40-60 grams 5.22 0.34 N.S. 4 60-80 grams 4.88 41 TABLE 6. The s i g n i f i c a n c e of differences (Keul's method) of mean per cent of hyaline amoebocytes i n blood between 20 gram wet weight groups of H. aurantium. Per cent data have been transformed by the arc sine method. Significance le v e l s are noted below mean differences in the table. X Angle X-4 X-3 X-2 WEIGHT GROUP No. Range 1 40-60 grams 42.14 10.54 4.63 0.01 .01 N.S. N.S. 2 60-80 grams 42.13 10.53 4.62 .01 N.S. 3 20-40 grams 37.51 5.91 .05 4 0-20 grams 31.60 42 TABLE 7. The r e l a t i v e concentration of blood c e l l types in the blood of H. aurantium as the mean per cent of p a r t i c u l a r c e l l type for each twenty gram wet weight group. Only those blood c e l l types which show a s i g n i f i c a n t difference i n r e l a t i v e concentration between wet weight groups are presented. WEIGHT GROUP 0-20 20-40 40-60 60-8( C e l l Type P E R C E N T Mature Morula 4.8 2.5 2.4 1.7 'Vacuolar' Types 35.2 31.7 27.0 27.4 D.V. C e l l 2.6 1.9 1.2 0.9 Stem C e l l 6.7 4.7 4.7 2.9 S.A.V.G. 1.8 0.9 0.9 0.5 Hyaline Amoebocytes 27.7 37.3 45.1 45.1 43 the matrix of the tunic. The epidermis i s attached firmly to a basement membrane. From the basement membrane connective sheets extend p e r i o d i c a l l y to bundles.of c i r c u l a r and l o n g i t u d i n a l muscles. The subepidermal blood space i s the c a v i t y between the epidermis and the muscle layers of the body wall through which the connective sheets extend (Fig. 4). (b) Tunic Matrix: The greatest portion of the tunic consists of. the fibrous matrix in which may be observed a number of blood c e l l s . The f i b e r s , which extrude from the a p i c a l ends of the epidermal c e l l s , coalesce just d i s t a l to the epidermis to form a fibrous layer or lamina. The matrix consists of a regular arrangement of these laminae which form successive sheets of tunic material around the entire animal. Individual lamina are joined together by fibrous extensions between them. In larger animals, the laminae are grouped into bands; within a band, laminae are approximately of the same dimensions, but the laminar dimensions between bands d i f f e r . Consequently, cross sections of tunic from larger animals appear to be made up of bands of thick and thin fibrous laminae (Fig. 4). There i s a p o s i t i v e c o r r e l a t i o n between number of laminae and wet weight of animal, c o r r e l a t i o n c o e f f i c i e n t (r) = + 0.95. The confidence l i m i t s in the calculated c o r r e l a t i o n c o e f f i c i e n t are 0.01. Linear regression analysis of the number of laminae as a function of wet weight demonstrates a slope of 1.668 and an intercept of 25.5. The p r o b a b i l i t y that t h i s slope value i s due to random p r o b a b i l i t y i s less than 0.001. Confidence l i m i t s at the 0.95 l e v e l for the estimation of number of laminae for a given weight have been calculated (Fig. 5). 44 FIGURE 4. The tunic of H. aurantium stained with Gomori's hematoxylin and phloxin. A. Cross section of body wall tunic. Sp, spine; La, laminae; Ma, matrix; Ep, epidermis. 150X. B. Cross section at the l e v e l of the epidermis. • SB, subepidermal blood space; Ep, epidermis; MM, mature morula c e l l s ; La, lamina. 800X. Note the aggregation of mature morula c e l l s j u s t above the epidermis, c. Cross section of a p a p i l l a and spine. Sp, spine; DV, dispersed v e s i c u l a r c e l l . 500X. 45 FIGURE 5. The number of laminae, or f i b r o u s l a y e r s , i n the t u n i c as a f u n c t i o n of whole animal wet weight i n grams. The s o l i d l i n e represents the c a l c u l a t e d l i n e a r r e g r e s s i o n l i n e . The dashed l i n e represents the 95% confidence l i m i t s i n the e s t i m a t i o n of a y value f o r any given x value. The c o r r e l a t i o n c o e f f i c i e n t i s + 0.95. 46 (c) Spinous Processes: The external surface of the tunic i s formed of p a p i l l a r outgrowths of the matrix upon which are situated c u t i c u l a r spines. Towards the periphery of the tunic the laminar structure of the matrix gradually i s l o s t , and the f i b e r s extend outwards forming a p a p i l l a (Fig. 4). The height of the p a p i l l a and i t s spine i s quite uniform (mean = 271 microns, n = 11). The spine i s approximately 160 microns in width (n = 11) at i t s base, which i s on top of the p a p i l l a , and tapers to an extremely f i n e point. The f i b e r s of the p a p i l l a pass up the core of the spine for some distance, but the spine i s hollow. Spines are c i r c u l a r i n o u t l i n e as i s the hollow core. Blood c e l l s occasionally may be seen in the hollow core of the spine. The material of the spine i s homogeneous and does not appear to have any microscopic substructure (Fig. 4). (d) Stolon: The hold-fast of H. aurantium consists of a number of stolons which are f i n g e r - l i k e outgrowths of the tunic, the underlying epidermis, and sub-epidermal blood spaces. The epidermis of the stolons i s more cuboidal than that of the body w a l l . The tunic matrix material of natural hold-fast stolons i s i r r e g u l a r and tunic p a p i l l a e and spines are absent, although a c u t i c u l a r condensation product i s evident (Fig. 6). In the laboratory, stolon material grows f r e e l y from the base of the animal. Over several months, these stolons w i l l reach approximately 1.0 mm. in length and between 0.1 and 0.25 mm. i n diameter. Tunic matrix of these stolons displays more r e g u l a r i t y of structure than do natural stolons. At the d i s t a l 47 t i p of the stolon, the matrix material i s quite th i n and ir r e g u l a r (Fig. 6 ) , but, as the body wall of the animal i s approached, the matrix becomes thicker and assumes a more regular and laminated appearance. The t i p of the stolon i s covered with an extremely thin c u t i c u l a r layer, but proximal, near the body of the animal, p a p i l l a and spines, in d i f f e r e n t stages of development, are evident. There i s a gradation in the development of lamination of f i b e r s , p a p i l l a , and spines from the d i s t a l t i p of the stolon to i t s junction with the body w a l l . At the t i p , p a p i l l a and spines are absent, and the fi b e r s of the tunic matrix are unorganized. As the body wall of the animal i s approached, the f i b e r s become more organized and s t a r t to assume a laminated appearance, and the p a p i l l a and spines acquire c h a r a c t e r i s t i c s of those found in body wall tunic. 5. Blood C e l l s in the Tunic: (a) Blood C e l l Concentration i n the Tunic: Four blood c e l l types are recognizable i n the tunic a f t e r s taining with chrome hemotoxylin-phloxin. They are the mature morula c e l l , dispersed v e s i c u l a r c e l l , hyaline amoebocyte, and granular amoebo-cyte. Comparisons (t-test) of mean c e l l concentration per cubic millimeter of body wall, or stolon, tunic versus blood concentration, demonstrate that a l l four c e l l types are at higher l e v e l s in the tunic (Table 8 ) . Comparisons of mean c e l l concentration i n tunic of the body wall versus tunic of the stolon show that the le v e l s of a l l four c e l l s are higher in the stolon than in the body wall (Table 8 ) . Within the 48 FIGURE 6. Laboratory grown stolon and injured body wall tunic of H. aurantium stained with Gomori's hemotoxylin with phloxin. A. Cross section of laboratory grown stolon near the growing t i p . SB, subepidermal blood space; MM, mature morula c e l l s ; Ma, matrix. 400X. Note the aggregation of mature morula c e l l s at the periphery of the tunic and lack of laminar structure of the matrix. B. Cross section of the external edge of the growing t i p of stolon. Ma, matrix; MM, mature morula c e l l , DV, dispersed v e s i c u l a r c e l l . 2,000X. C. Cross section of tunic f i v e days a f t e r injury. LI, l i m i t s of i n c i s i o n ; LC, coalescence and c o n s t r i c t i o n of laminae at wound edge; MD, mature morula degradation products; MM, mature morula c e l l s . 200X. D. Cross section of tunic f i v e days aft e r injury. MD, mature morula degradation products; LI, external l i m i t s of the i n c i s i o n . The epidermis i s to the bottom of the p i c t u r e . Note the dark staining mature morula degradation products throughout the wound area, and the c o n s t r i c t i o n of laminae to the sides of the wound. 100X. 49 TABLE 8. The concentration of blood c e l l s per cubic millimeter (x 10) in blood and tunic with s i g n i f i c a n c e l e v e l s (P) of difference in concentration based on Student's t - t e s t . Tunic indicates tunic on the body wall, t u n i c s indicates tunic of the stolon. B Mature Morula D.V.Cell Hyaline Granular Blood Mean S.E. 0.54 0.10 0.38 0.09 3.14 0. 58 2.26 0.42 T u n i c B Mean S.E. 2.84 0.66 11.06 1.55 6.43 0.89 6.62 0.76 Tunic s Mean S.E. 123.18 23.99 47.50 9.62 45.32 8.96 47.50 9.62 Blood vs T u n i c B P 0.02 0.001 0.02 0.001 Blood vs T u n i c s P 0.01 0.01 0.001 0.01 Tunic vs B Tunicg P 0.01 0.01 0.01 0.01 50 t u n i c o f the body w a l l , the matura morula and d i s p e r s e d v e s i c u l a r c e l l show d i s c r e t e p o s i t i o n a l r e l a t i o n s h i p s . Based on a sample o f f i v e animals, the mature morula c e l l i s co n c e n t r a t e d a d j a c e n t to the epidermis, whereas the d i s p e r s e d v e s i c u l a r c e l l i s c o n c e n t r a t e d a t the e x t e r n a l l i m i t s o f the t u n i c . Mature morula c e l l s are r a r e i n the out e r p o r t i o n o f the t u n i c , but the d i s p e r s e d v e s i c u l a r c e l l i s evident, i n s m a l l numbers, throughout the t u n i c ( F i g . 7). The h y a l i n e amoebocyte and g r a n u l a r amoebocyte do not show any p a r t i c u l a r p o s i t i o n a l r e l a t i o n s h i p s i n the t u n i c , b ut appear to be s c a t t e r e d randomly. I f the c o n c e n t r a t i o n o f mature morula and d i s p e r s e d v e s i c u l a r c e l l s i n the i n n e r and out e r h a l v e s o f the t u n i c are compared f o r both body w a l l t u n i c and s t o l o n , measured a t the h a l f d i s t a n c e between growing t i p and body w a l l , t h e r e i s a s i g n i f i c a n t l y h i g h e r c o n c e n t r a t i o n o f mature morula c e l l s i n the i n n e r h a l f o f the t u n i c a d j a c e n t to the epidermis, and a s i g n i f i c a n t l y h i g h e r c o n c e n t r a t i o n o f d i s p e r s e d v e s i c u l a r c e l l s i n the out e r o r e x t e r n a l h a l f o f the t u n i c (Table 12). At the growing t i p o f l a b o r a t o r y grown s t o l o n , the t u n i c m a t e r i a l i s q u i t e t h i n and t h e r e i s no p o s i t i o n a l s e p a r a t i o n o f mature morula and d i s p e r s e d v e s i c u l a r c e l l s . Both the mature morula and d i s p e r s e d v e s i c u l a r c e l l undergo m o r p h o l o g i c a l changes i n the t u n i c . The mature morula c e l l breaks down i n the t u n i c , e i t h e r by a d i s p e r s i o n o f i t s vac u o l e s , as i f the c e l l membrane had been d i s r u p t e d , o r by a coa l e s c e n c e o f the s m a l l e r v a c u o l e s i n t o one or two l a r g e r v a c u o l e s which appear to be empty. Mature morula c e l l s which have undergone these changes i n the t u n i c are r e f e r r e d t o as t u n i c v e s i c u l a r c e l l s and have no c o u n t e r p a r t i n the b l o o d . 51 FIGURE 7. The concentration, in thousands of c e l l s per cubic millimeter of mature morula and D.V. c e l l s as a function of p o s i t i o n in tunic. F i e l d 1 represents the f i e l d adjacent to the epidermis, and f i e l d 5 is at the periphery of the tunic. i 45 35 CO 25 co _ i L L . O g 15 10 0 MATURE MORULA CELL D. V. CELL fed ,rVf I •3 3 -21 -1 2 3 F I E L D FROM EPIDERMIS The dispersed v e s i c u l a r c e l l , once i t has reached the outer portions of the tunic, displays more subtle changes. The v e s i c l e s of t h i s c e l l appear to be smaller and are more d i f f i c u l t to s t a i n . The cytoplasm around the v e s i c l e s has dissipated and w i l l not s t a i n . The nucleus assumes a necrotic appearance. 6. Blood C e l l Function i n the Tunic: (a) Phagocytosis: The hyaline amoebocytes were the only c e l l s which had i n t r a c e l l u l a r carbon p a r t i c l e s f o r t y - e i g h t hours a f t e r the i n j e c t i o n of a carbon suspension into the tunic of H. aurantium. Analysis of variance of the mean per cent of hyaline amoebocytes with carbon p a r t i c l e s as a function of time a f t e r carbon i n j e c t i o n (0, 3, 6, 12, 18, 24 and 48 hours) displays a s i g n i f i c a n t F r a t i o (P < 0.01). There i s an increase i n r e l a t i v e numbers of hyaline amoebocytes with carbon with time a f t e r i n j e c t i o n (Fig. 8) with the maximum of approximately 45% reached a f t e r eighteen hours. Comparsions of the mean per cent of hyaline amoebocytes with carbon p a r t i c l e s for each time period and the l e v e l s of s i g n i f i c a n t differences between means are given i n Table 9. (b) Blood C e l l s i n Injured Tunic: Analyses of variance of the concentration per cubic millimeter of the four blood c e l l s in the tunic as a function of day a f t e r injury reveal s i g n i f i c a n t increases in the number of mature morula, including the tunic v e s i c u l a r c e l l (P < 0.005), and the dispersed v e s i c u l a r c e l l s (P < 0.005). The maximum concentration of these two c e l l types i s reached by the f i f t h day a f t e r injury (Fig. 9). By the t h i r d day a f t e r injury, there i s a s i g n i f i c a n t l y higher concen-t r a t i o n of mature morula c e l l s i n the i n j u r y area than there i s i n control preparations (Table 10). By the f i f t h day, the concen-t r a t i o n of dispersed v e s i c u l a r c e l l s i s s i g n i f i c a n t l y higher than i n controls (Table 11). F i f t e e n days a f t e r injury there i s no 53 FIGURE 8. The per cent of hyaline amoebocytes with carbon p a r t i c l e s as a function of time a f t e r carbon i n j e c t i o n into the tunic. Per cent data have been transformed by the arc sine method. 0 3 6 12 18 24 HOURS A F T E R CARBON 48 NJECTION 54 TABLE 9. The l e v e l s of s i g n i f i c a n c e o f mean d i f f e r e n c e s i n the p e r cent o f h y a l i n e amoebocytes with carbon p a r t i c l e s as a f u n c t i o n o f time a f t e r carbon i n j e c t i o n . The per cent d a t a have been transformed by the a r c s i n e method. The l e v e l s o f s i g n i f i c a n c e are noted below the mean d i f f e r e n c e s i n the body of the t a b l e . The mean per cent o f c e l l s w i t h carbon are noted, i n parentheses, below the mean angle. X Angle X-0 Time Hours 18 42.63 28.53 (45.9%) 0.01 12 41.71 27.61 (44.4%) 0.01 48 40.18 26.08 (41.8%) 0.01 24 39.57 25.47 (40.8%) 0.01 6 31.52 17.42 (27.8%) 0.05 3 20.90 6.80 (13.3%) N.S. X-3 X-6 X-24 X-48 21.73 0.05 11.11 N.S. 3.06 N.S. 2.45 N.S. 20.81 0.05 10.19 N.S. 2.14 N.S. 1.63 N.S. 19.28 0.05 8.66 N.S. 0.61 N.S. 18.67 0.05 8.05 N.S. 10.62 N.S. 0 14.10 (6.5%) 5 5 FIGURE 9. The concentration of mature morula c e l l s , including tunic v e s i c u l a r c e l l s , and dispersed v e s i c u l a r c e l l s (x 1C-3) per cubic millimeter of tunic as a function of day afte r injury. The ordinate on the r i g h t refers to the mature morula c e l l s , and on the l e f t to the dispersed v e s i c u l a r c e l l s . NUMBER CELLS/MM 3 x10 3 IN TUNIC (D .V .CELLS) o o NUMBER C E L L S / M M 3 x 10 3 r-o 4N cn IN°TUNIC C (MJVI.CELLS) CO o r o o o 56 TABLE 10. The s i g n i f i c a n c e of mean differences i n the number of mature morula c e l l s (x 10 3) , including tunic v e s i c u l a r c e l l s , per cubic millimeter of tunic as a function of day a f t e r injury. DAY X _ Cells/mm 3 X-0 X - l X-15 X-10 X-3 5 191.2 170.9 118.4 101.2 68.9 56.9 0.01 0.05 0.05 N.S. N.S, 3 134.3 114.0 61.5 44.3 12.0 0.05 N.S. N.S. N.S. 10 122.3 102.0 49.5 32.2 0.05 N.S. N.S. 15 90.0 69.7 17.2 N.S. N.S. 1 72.8 52.5 N.S. 0 20.3 57 TABLE 11. The s i g n i f i c a n c e of mean differences in the number of D.V. C e l l s (x 10^) per cubic m i l l i -meter of tunic as a function of day a f t e r i n j u r y . DAY X Cells/mm 3 X-0 X - l X-3 X-15 X-10 5 27.1 22.2 21.4 9.5 4.0 2.2 0.05 0.05 N.S. N.S. N.S. 10 24.9 20.0 19.2 7.3 1.8 0.05 0.05 N.S. N.S. 15 23.1 18.2 17.4 5.5 0.05 0.05 N.S. 3 17.6 12.7 11.9 N.S. N.S. 1 5.7 0.8 N.S. 0 4.9 58 s i g n i f i c a n t difference in the concentration of dispersed v e s i c u l a r c e l l s from t h e i r maximum l e v e l at f i v e days a f t e r injury. By the f i f t e e n t h day, however, the mature morula c e l l has s i g n i f i c a n t l y decreased from t h e i r maximum concentration at the f i f t h day. The hyaline amoebocytes and granular amoebocytes do not increase s i g n i f i c a n t l y in the injury area over the measured time period (15 days). In uninjured tunic, the greatest concentration of mature morula c e l l s i s in the inner h a l f of the tunic adjacent to the epidermis. In the case of the dispersed v e s i c u l a r c e l l s , the greatest concentration i s at the peripheral l i m i t s of the tunic. The e f f e c t of injury to the tunic i s to abolish t h i s p o s i t i o n a l r e l a t i o n s h i p . Within a day after injury, although there are higher l e v e l s of both c e l l types i n the tunic, the c e l l s are equally d i s t r i b u t e d between the inner and outer halves of the tunic in the injury area. This i s not a permanent s i t u a t i o n . By the tenth day af t e r injury, there are s i g n i f i c a n t l y higher concentrations of dispersed v e s i c u l a r c e l l s i n the outer h a l f of the tunic. By the f i f t e e n t h day af t e r injury, there are s i g n i f i c a n t l y higher concentrations of mature morula c e l l s i n the inner h a l f of the tunic (Table 12). By the f i f t e e n t h day, the p o s i t i o n a l relationships of these two c e l l types are re-established i n the tunic. (c) Morphology of the Injury Area: As the mature morula and dispersed v e s i c u l a r c e l l migrate into the tunic, there are p a r t i c u l a r changes i n both these c e l l types and the tunic tissue which they invade (Fig. 6). The mature morula c e l l breaks down into tunic v e s i c u l a r c e l l s throughout the injury area. I n i t i a l l y , TABLE 12. The s i g n i f i c a n c e l e v e l s of the differences in mean concentration of D.V. and Mature Morula c e l l s per cubic millimeter between the inner (+) and outer (-) halves of the tunic as a function of time a f t e r injury. Also included i n the table, are the si g n i f i c a n c e l e v e l s of the differences in concentration of these c e l l types in si m i l a r areas of uninjured and stolon tunic. C e l l Type Mature Morula D.V. C e l l Day a f t e r Injury Stolon +0.01 -0.001 Uninjured Tunic +0.025 . -0.005 0 +0.001 N.S. 1 N.S. N.S. 3 N.S. N.S. 5 N.S. N.S. 1 0 N.S. -0.01 1 5 +0.01 -0.001 i t appears t o congregate and break down a t the edge o f the wound. The l a m i n a t i o n o f the t u n i c matrix, which has been cut by the i n i t i a l i n c i s i o n , are pinch e d and p u l l e d t o g e t h e r ( F i g . 6) . At t h e edge of the wound, the volume o f the d i s p e r s e d v e s i c u l a r c e l l s i n c r e a s e s , the nucleus assumes a n e c r o t i c appearance, and the v e s i c l e s decrease i n diameter. Twenty days a f t e r i n j u r y , the wound area shows a s l i g h t accumulation o f randomly o r i e n t e d f i b r o u s m a t e r i a l which s t a i n s l i k e the t u n i c f i b e r s o f the m a t r i x . The e x t e r n a l edge o f the wound, by the t w e n t i e t h day, i s covered with a t h i n d e n s e l y s t a i n i n g c u t i c u l a r m a t e r i a l , which i s r e m i n i s c e n t o f the m a t e r i a l c o v e r i n g the i n t e r p a p i l l a r y areas o f u n i n j u r e d t u n i c . The f i b r o u s m a t e r i a l below the wound i s l i f t e d towards the i n j u r y and c o n s t r i c t s t h e r e . The wound i t s e l f upon macroscopic examination appears more dense and rough i n t e x t u r e than u n i n j u r e d t u n i c . T h i s area i s not as h e a v i l y pigmented as normal t u n i c , although i t does d i s p l a y a s l i g h t y e l l o w i s h c o l o r a t i o n . In some i n j u r e d animals, which were not i n c l u d e d i n the c e l l m i g r a t i o n s t u d i e s , the epidermis as w e l l as the t u n i c was i n j u r e d . Macroscopic examination a f t e r a t h i r t y day p e r i o d r e v e a l s t h a t no apparent epidermis p r o l i f e r a t e s over the wound are a . There i s no p r o l i f e r a t i o n over the wound o f t u n i c m a t e r i a l , although the edges o f the wound are rough and r a i s e d and appear to be s i m i l a r t o i n j u r e d t u n i c o f animals i n which the epidermis was not damaged. There was no c l o s u r e o f the wound by t u n i c m a t e r i a l over t h i s t h i r t y day p e r i o d . 61 7 . Histochemistry of the Blood C e l l s : (a) Histochemistry of the Blood C e l l s i n C i r c u l a t i o n : Conventional methods of sta i n i n g for neutral and acid mucopoly-saccharides indicate that the vacuolar contents of the mature morula (Talbe 14), immature morula, compartment c e l l , and signet r i n g c e l l s do not contain these substances. The cytoplasm of these c e l l s , however, w i l l s t a i n occasionally by the p e r i o d i c acid S c h i f f method. The hyaline amoebocytes and the granular amoebocytes show uniformly the presence of some P.A.S. p o s i t i v e material. In these cases, the P.A.S. p o s i t i v e material i s either d i f f u s e , in the hyaline amoebocytes, or granular in the granular amoebocyte. Hyaline amoebocytes dis p l a y an occasional p o s i t i v e orthochromatic reaction to to l u i d i n e blue (Table 13). Vesi c l e s of the dispersed v e s i c u l a r c e l l w i l l react p o s i t i v e l y with P.A.S. only i f they have been fixed with cyanuric chloride and N-methyl morpholine. When prepared with other f i x a t i v e s , the v e s i c l e s of t h i s c e l l are ambiguous i n t h e i r reaction to P.A.S. Vesi c l e s of the dispersed v e s i c u l a r c e l l are also p o s i t i v e to c o l l o i d a l iron, and show a range, from p o s i t i v e to negative between c e l l s , of reaction to a l c i a n blue and aldehyde fuchsin. No observations for the reaction of the stem c e l l and stem c e l l with a c i d o p h i l i c vacuoles or granules to P.A.S. are av a i l a b l e . The stem c e l l shows, however, a p o s i t i v e orthochromatic reaction to to l u i d i n e blue and azure A at pH 1.5. The stem with a c i d o p h i l i c vacules or granules i s negative to azure A at pH 1.5, but displays a p o s i t i v e reaction to aldehyde fuchsin. Blood c e l l s , i n general, d i s p l a y a f a i n t uniform p o s i t i v e reaction to M i l l o n reagent. This reaction i s r e s t r i c t e d to the cytoplasm and there i s no concentration of a M i l l o n p o s i t i v e material i n the vacuoles, v e s i c l e s , or granules. Immature morula c e l l s d isplay a p o s i t i v e reaction to Biebrich's s c a r l e t , whereas the mature morula, compartment, and signet r i n g ' c e l l s d i s p l a y a range of reactions from s l i g h t l y p o s i t i v e to negative. The hyaline amoebocyte, the granular amoebocyte, the stem c e l l and the stem with a c i d o p h i l i c vacuoles or granules are a l l negative to Biebrich's s c a r l e t . The mature morula, the immature morula, the compartment, and the signet r i n g c e l l s a l l s t a i n an intense v i o l e t with mercuric bromophenol blue. The ve s i c l e s of the dispersed v e s i c u l a r c e l l are p o s i t i v e to Biebrich's s c a r l e t and mercuric bromophenol blue. The reaction of the remaining c e l l types to Biebrich's s c a r l e t and mercuric bromophenol blue ranges from f a i n t to negative. A l l of the blood c e l l s d i s p l a y a negative reaction to the DDD sulfhydryl technique. Vacuoles of the mature morula, immature morula, compartment, and signet ri n g c e l l s show a methylene blue extinction point at pH 9.05. The extincti o n point of the hyaline and granular amoebocytes i s pH 5.00 and that of the stem c e l l i s pH 4.00. R.N.A., as indicated by RNase l a b i l i t y , i s present d e f i n i t e l y i n the stem c e l l and stem c e l l with a c i d o p h i l i c vacuoles or granules. Hyaline amoebocytes and signet r i n g c e l l s o ccasionally demonstrate R.N.A. The mature morula, immature morula, and compartment c e l l s t a i n negative for R.N.A. The dispersed v e s i c u l a r c e l l displays a v a r i e t y of conditions; some of these c e l l s d e f i n i t e l y show R.N.A., but others are completely negative. None of the blood c e l l s s t a i n for the presence of iron unless they are pretreated with o x a l i c acid and hydrogen peroxide. Afte r treatment, the mature morula, immature morula, compartment c e l l , and the signet ri n g c e l l do st a i n for iron. Other blood c e l l s do not demonstrate the presence of iron under any of the conditions attempted. The histochemical reactions of the hyaline amoebocyte, granular amoebocyte, the mature morula c e l l , and the dispersed v e s i c u l a r c e l l in blood are summarized in Tables 13 and 14. (b) Histochemistry of the Mature Morula C e l l and Dispersed Vesicular C e l l i n Tunic: The mature morula c e l l , in the tunic, retains i t s general negative reaction to poly-saccharide s t a i n s . I t has been noted, however, in numerous preparations that the v e s i c l e s within a mature morula c e l l demonstrate a v a r i e t y of staining reactions: with azure A at pH 4.0 the reaction of the v e s i c l e s ranges from negative to intensely p o s i t i v e , with aldehyde fuchsin from f a i n t to d e f i n i t e l y p o s i t i v e , and with t o l u i d i n e blue from f a i n t l y p o s i t i v e to negative. In other mature morula c e l l s in the same preparations', a l l the v e s i c l e s within a given c e l l are either p o s i t i v e or negative to aldehyde fuchsin and azure A at pH 4.0. The v e s i c l e s of the dispersed v e s i c u l a r c e l l r e t a i n t h e i r p o s i t i v e reaction to the P.A.S. technique when fixed with cyanuric chloride and N-methylmorpholine, but these v e s i c l e s are negative to a l l other polysaccharide stains when the c e l l s are at the periphery of the tunic. The sta i n i n g reactions of the dispersed v e s i c u l a r and mature morula c e l l in the tunic are 64 TABLE 13. The histochemical reactions of the hyaline amoebocyte and granular amoebocyte in the blood of aurantium. The reaction of the c e l l s are indicated as follows: + d e f i n i t e l y p o s i t i v e , + f a i n t l y p o s i t i v e , - negative. Alpha refers to an orthochromatic reaction. Hyaline Granular Amoebocyte Amoebocyte REACTION P.A.S. + + Alc i a n Blue Toluidine Blue + (alpha) Aldehyde Fuchsin - + to -Methylene Blue E x t i n c t i o n (pH) 5.00 5.00 Methyl Green Pyronin (R.N.A.) + to - + Perl's Iron (H 20 2? Oxalate) Millon's Rgt. + + TABLE 14. The histochemical reactions of the mature morula, D.V. c e l l , and tunic v e s i c u l a r c e l l s in both the blood and tunic are summarized. The following convention was u t i l i z e d to express i n t e n s i t y of stai n i n g ; - negative, +_ f a i n t p o s i t i v e , + d e f i n i t e l y p o s i t i v e , ++ strong p o s i t i v e , and +++ intense p o s i t i v e . Alpha refers to an alpha metachromasia or orthochromasia. REACTION MATURE MORULA Blood Tunic VESICULAR Tunic D.V. CELL Blood Tunic P.A.S. Alcia n Blue Toluidine Blue C o l l o i d a l Iron Azure A pH 1.5 Azure A pH 4.0 Aldehyde Fuchsin + + + to -(alpha) + to -++ to + ++ to + + + '+ to -+ + ++ to + to -Methylene Blue Ext i n c t i o n 9.05 (pH) Methyl Green Pyronin (RNA) DDD Sulfhydryl Biebrich's Scarlet + to -9.05 N.R. + to -5.00 + to 5.35 + Mercury Bromo-phenol Blue + Millon's Rgt. + Perl's Iron (H 20 2: Oxalate) + to + + + N.R. + + + 6 6 summarized in Table 14. The v e s i c l e s of the mature morula c e l l s are f a i n t l y p o s i t i v e to negative with Biebrich's s c a r l e t and p o s i t i v e to mercuric bromophenol blue in the tunic. The v e s i c l e s are negative for sulfhydryls according to the DDD reaction. The dispersed v e s i c u l a r c e l l i s negative to a l l three of these stains in the tunic, as well as for R.N.A. After treatment with o x a l i c acid and hydrogen peroxide, the mature morula c e l l s in the tunic display a s l i g h t p o s i t i v e reaction for iron. The dispersed v e s i c u l a r c e l l in the tunic does not show iro n . The large condensation vacuoles of the tunic v e s i c u l a r c e l l s , the mature morula break down product in the tunic, appear to be empty upon morphological staining and only occasionally do the vacuolar contents take a histochemical s t a i n . Rarely, the vacuole w i l l show a d e f i n i t e l y p o s i t i v e to strongly p o s i t i v e reaction to aldehyde fuchsin, a f a i n t reaction to Biebrich's s c a r l e t , or a d e f i n i t e l y p o s i t i v e to negative reaction to Hale's c o l l o i d a l iron (Table 14). 8. Histochemistry of the Tunic: (a) Histochemistry of the Epidermis: The epidermis displays a granulation which i s both P.A.S. and Hale's c o l l o i d a l i r o n p o s i t i v e . The epidermis also stains intensely with aldehyde fuchsin, a l c i a n blue, and azure A at pH 1.5 and 4.0. The epidermis shows a d e f i n i t e beta metachromasia with t o l u i d i n e blue. After 24 hours methylation, the epidermis w i l l not stain with either azure A at pH 1.5 or aldehyde fuchsin. There i s a decrease i n int e n s i t y of staining with azure A at pH 4.0, al c i a n blue, and Hale's c o l l o i d a l iron. Upon saponification of methylated tissue, the epidermis shows an increase in staining i n t e n s i t y over methylated tissue with a l l the above stains except a l c i a n blue and Hale's c o l l o i d a l iron. In no case does the staining i n t e n s i t y of methylated and saponified epidermis return to the l e v e l of untreated t i s s u e . The epidermis shows a weak, but p o s i t i v e reaction, to Mi l l o n reagent, DDD sul f h y d r y l technique, and Biebrich's s c a r l e t . I t i s generally negative i n i t s reaction to mercuric bromophenol blue. The epidermis displays a strong p o s i t i v e reaction for the presence of R.N.A. based on the methyl green pyronin technique with RNase controls. The methylene blue extinction point of the epidermis i s below pH 1.63. (b) Histochemistry of the Tunic Matrix: The tunic matrix f i b e r s give a f a i n t to negative reaction to the P.A.S. technique but are intensely p o s i t i v e to aldehyde fuchsin, a l c i a n blue, and Hale's c o l l o i d a l iron. The fi b e r s also give an intense p o s i t i v e reaction to azure A, at pH's 1.5 and 4.0, and to l u i d i n e blue with a br i g h t gamma metachromasia (red). Upon methylation for 24 hours there i s a marked decrease in sta i n i n g reaction with both azure A's, aldehyde fuchsin, a l c i a n blue, and c o l l o i d a l iron. Methylation followed by saponif i c a t i o n r e s u l t s i n an increase i n sta i n i n g i n t e n s i t y with azure A at pH 1.5 and 4.0, but not to the l e v e l of untreated tissue. This type of treatment does not cause any increase in staining i n t e n s i t y from the methylated l e v e l with aldehyde fuchsin, a l c i a n blue, and c o l l o i d a l iron. The methylene blue extinction point of tunic f i b e r s i s below pH 1.63. The tunic f i b e r s 68 di s p l a y an extremely f a i n t to negative reaction with M i l l o n reagent or the DDD sul f h y d r y l reaction. These f i b e r s are, however, d e f i n i t e l y p o s i t i v e to Biebrich's s c a r l e t and display a f a i n t to negative reaction with mercury bromophenol blue. The tunic f i b e r s do not display either R.N.A. or iron by the methods employed. (c) Histochemistry of the Tunic Spines: The tunic spines are negative to P.A.S., a l c i a n blue, and Hale's c o l l o i d a l iron. These structures show an extremely f a i n t p o s i t i v e reaction to azure A at pH 1.5 and a d e f i n i t e l y p o s i t i v e reaction to azure A at pH 4.0. They are f a i n t l y p o s i t i v e to aldehyde fuchsin and are orthochromatically p o s i t i v e to to l u i d i n e blue. The staining i n t e n s i t y of the spines with azure A at pH 4.0 does not seem to be affected by either methylation or saponif i c a t i o n . The spines are negative in reaction to the DDD sulfhydryl test, extremely f a i n t p o s i t i v e to Millon's reagent, negative to Biebrich's s c a r l e t , and d e f i n i t e l y p o s i t i v e to mercury bromophenol blue. The l a s t reaction i s p a r t i c u l a r l y intense at the external edges of the spines. The spines do not display the presence of either iron or R.N.A. A summary of the h i s t o -chemical reactions of the three tunic components i s given i n Table 15. 9. Enzymatic Analyses of Tunic and Tunicin: (a) Pronase Digestion of Tunic: Treatment of tunic with pronase for 48 hours at 37°C re s u l t s in a loss of 30.9% (n = 3, S.E. 1.0%) of the o r i g i n a l dry weight (Table 17). (b) C e l l u l a s e Digestion of Tunic and Tunicin: C e l l u l a s e 69 TABLE 15 REACTION The histochemical reactions of the three tunic components, matrix f i b e r s , spines, and epidermis. The following convention was used to indicate i n t e n s i t y of staining: - negative, + f a i n t p o s i t i v e , + d e f i n i t e l y p o s i t i v e , ++ strong p o s i t i v e , +++ intense p o s i t i v e . Alpha, beta, and gamma re f e r to orthochromatic color, v i o l e t , and red metachromasia respectively. Matrix Fibers Spines Epidermis P. A. S. +_ to -Methylene Blue pH 1.63 Toluidine Blue +++ gamma Azure A pH 1.5 +++ Methylated + to -Saponified ++ to + Azure A pH 4.0 +++ Methylated + Saponified ++ Aldehyde Fuchsin +++ Methylated ++ Saponified ++ Al c i a n Blue +++ Methylated ++ to + Saponified + C o l l o i d a l Iron +++ Methylated ++. to + Saponified ++ to + DDD Sulfhydryl Biebrich's Scarlet + Millon's Rgt. + Mercuric Bromo-phenol Blue +_ to -Methyl Green Pyronin (RNA) pH 4.28 + alpha + + + + + -f + pH 1.63 + beta ++ + ++ + to +_ + ++ + + + + + + + + + to -+ 70 does not y i e l d measurable r e d u c i n g sugar from t u n i c a f t e r 14 days o f i n c u b a t i o n w i t h the enzyme. D i g e s t i o n o f t u n i c , which has been p r e - t r e a t e d w i t h pronase f o r 48 hours, does not y i e l d measurable amounts o f r e d u c i n g sugar over a 28 hour p e r i o d . However, i f pronase t r e a t e d t u n i c i s swollen w i t h 85% p h o s p h o r i c a c i d f o r 8 hours p r i o r t o the c e l l u l a s e d i g e s t i o n o f 48 hours, then 12.1% (n = 3, S.E. = 0.3%) o f the d r y weight o f the t u n i c i s r e l e a s e d as r e d u c i n g sugar ( P i g . 10). F u r t h e r s w e l l i n g and i n c u b a t i o n o f t h i s t u n i c w i t h f r e s h c e l l u l a s e b e f o r e a 24 hour and then b e f o r e a 48 hour i n c u b a t i o n p e r i o d produces another 7.2% o f the d r y weight as r e d u c i n g sugar. The t o t a l amount o f r e d u c i n g sugar o b t a i n e d from pronase t r e a t e d t u n i c by t h i s method i s 19.3% o f o r i g i n a l dry weight a f t e r 148 hours of i n c u b a t i o n w i t h c e l l u l a s e and t h r e e s w e l l i n g t r e a t -ments w i t h p h o s p h o r i c a c i d . T u n i c i n y i e l d s 12.7% (n = 3, S.E. = 0.4%) of i t s dry weight as r e d u c i n g sugar a f t e r 28 hours i n c u b a t i o n i n c e l l u l a s e . A f t e r t h i s t u n i c i n i s t r e a t e d w i t h p h o s p h o r i c a c i d , the y i e l d o f r e d u c i n g sugar i s 20.8% (n = 3, S.E. = 3.3%) of the d r y weight over 48 hours o f i n c u b a t i o n ( F i g . 10). Subsequent r e -s w e l l i n g and i n c u b a t i o n i n f r e s h c e l l u l a s e f o r a 24 and a 48 hour p e r i o d , r e s u l t s i n a t o t a l y i e l d o f 42.9% o f the d r y weight as. r e d u c i n g sugar f o r a t o t a l i n c u b a t i o n time o f 148 hours. The maximum y i e l d o f r e d u c i n g sugar o b t a i n e d from p h o s p h o r i c a c i d s w o l l e n t u n i c i n i s 55.9% (n = 4, S.E. = 2 . 6 % ) a f t e r 14 days c e l l u l a s e i n c u b a t i o n . The r e d u c i n g sugar o b t a i n e d from the 14 day c e l l u l a s e i n c u b a t i o n o f t u n i c i n g i v e s the same r f . v a l u e FIGURE 10. The e f f e c t of phosphoric acid treatment on the y i e l d of reducing sugar from t u n i c i n and pronase digested tunic upon c e l l u l a s e digestion. 0 4 8 12 16 20 24 28 32 36 40 44 48 HOURS INCUBATION IN C E L L U L A S E 72 as D-glucose standards run concurrently on t h i n layer chromotography plates. (c) Chitinase and Hyaluronidase Digestion of Tunic: Both tunic and pronase treated tunic are not susceptible to chitinase digestion over a 14 day period as measured by released reducing sugar and hexosamine. Similarly, hyaluronidase, as measured by released reducing sugar and hyaluronate, does not digest tunic a f t e r 14 days of incubation. (d) The E f f e c t of P r o t e o l y t i c Enzymes and Hyaluronidase on Tunic Morphology and Histochemistry: The e f f e c t of hyaluronidase, pronase, and trypsin/papain digestion of tunic structures i s summarized in Table 16. The epidermis of tunic, u t i l i z e d for enzyme studies, i s destroyed by the drying procedures. Morphologically, there does not appear to be any change in matrix structure as a r e s u l t of either p r o t e o l y t i c enzyme or hyaluronidase digestion. The laminae of the matrix r e t a i n t h e i r ordered arrangement within the tunic and are unchanged in appearance. The spines are eroded by the p r o t e o l y t i c enzymes. Degradation i s not complete, however, although i n many cases only traces of spinous processes remain. Histochemically, treatment of the tunic does not increase the P.A.S. s e n s i t i v i t y of the matrix f i b e r s or spines. Both of these systems remain negative i n t h e i r reaction to P.A.S. Alc i a n blue staining on the other hand i s diminished, but s t i l l of strong i n t e n s i t y . 10. Chemical Analyses of Tunic and Tunicin: (a) General: Tunicin, placed in Schweitzer's reagent, 73 TABLE 16. The ef f e c t s of various experimental treatments on the structure and Alci a n Blue-P.A.S. r e a c t i v i t y of the tunic f i b e r s and spines. The following abbreviations are used: A.B. = a l c i a n blue, P.A.S.= Periodic Acid S c h i f f reaction, Hy'ase = hyaluronidase, N.C. = no change i n the structure, N.R. = no reaction, CD. = completely degraded, and P.D. = p a r t i a l l y degraded. TUNIC FIBERS A.B. P.A.S. Remarks TUNIC SPINES A.B. P.A.S. Remarks TREATMENT Control +++ + to - N.C. N.C. Tunicin Prep. N.C. N.R. N.R. CD. 6N HC1 8 hours + Hyaluronidase ++ N.C. N.C. N.R. N.R. CD. P.D. Pronase/ Hy 1ase ++ N.C. P.D. Trypsin/ Papain ++ N.C. P.D. 74 hot concentrated l i t h i u m thiocyanate, or acetic anhydride at room temperature, does not swell or disperse over a 48 hour period. Tunicin swells, however, in 85% phosphoric acid at 10°C by eight hours. Attempts to acetylate t u n i c i n are unsuccessful. Upon addition of the a c e t o l y s i s reagent and heating, a thick t a r r y sludge i s formed. The water content of fresh tunic i s approximately 81% of the wet weight. The weight loss of fresh tunic upon drying with organic solvents i s 81.1% of the o r i g i n a l weight. Oven drying for 48 hours r e s u l t s i n a weight loss of 81.5%. Treatment of dry tunic with acid or base at high temperatures (ca. 100°C), or with base for extended periods of time (7 days) at room temperature, r e s u l t s in a weight loss of approximately 50% of the dry weight of tunic (Table 17). Tunicin preparation also displays a loss in weight of the o r i g i n a l tunic in t h i s range (Table 17). Pronase treatment of dry tunic y i e l d s a weight loss of approximately 31% (Table 17). (b) T r a n s i t i o n Metal Analyses: Colorimetric analyses for iron reveal that the blood c e l l s have the highest concentration r e l a t i v e to dry weight. Tunic, branchial sac, gut, and gonad display decreasing l e v e l s of iron i n that order (Table 18). The iron, in the blood c e l l s , i s in a non-d i a l y z a b l e f r a c t i o n of the cytolyzate. Iron assays by the colorimetric method (o-phenanthroline) show great v a r i a t i o n between animals and between the tissues of d i f f e r e n t animals. Spectrographic analyses of pooled blood c e l l s from f i v e animals TABLE 17. The r e l a t i v e loss in dry weight of tunic upon various treatments. TREATMENT PER CENT WEIGHT LOSS X % S . E . 6N HC1 47.1 1.7 4 hours; 100°C 6N HC1 52.0 3.6 8 hours; 100°C 4% NaOH 41.8 0.5 7 days; 20°C TUNICIN 49.4 0.8 Preparation Pronase 30.9 0.3 48 hours; 37°C 76 TABLE 18. The d i s t r i b u t i o n of iron in various tissues of H. aurantium. Iron content i s expressed as micrograms of iron per gram dry weight of tis s u e . Iron was assayed by the o-phenanthroline colorimetric method. Branchial Blood Whole Tunic Gonad Sac Gut C e l l s Animal Mean 112.2 30.3 42.2 42.0 127.0 117.4 S.E. 44.3 12.1 14.7 9.7 17.3 18.9 n 39 77 reveal a mean iron concentration of 407 p.p.m. dry weight (n = 3, S.E. = 23 p.p.m.). Titanium and manganese are present also at concentrations of 47 p.p.m. (n = 3, S.E. = 27 p.p.m.) and 17 p.p.m. (n = 3, S.E. = 2 p.p.m.) respectively. Vanadium, columbium, and chromium, i f present, are below the l i m i t s of detection by t h i s method. (c) Pigmentation: The tunic of H. aurantium i s pigmented heavil y . The tunic ranges from cream yellow to a dark peach in color. This pigment i s methanol extractable and, upon extraction of the methanol with n-hexane under N , remains in 2 the a l c o h o l i c phase. The blood does not display a d e f i n i t e pigment c e l l , but the hyaline amoebocytes occasionally show a large, bright yellow, granular i n c l u s i o n . A methanol soluble pigment i s extractable from blood c e l l s also. The absorption spectra of methanol extracts of tunic and blood c e l l s display coincident maxima at 445 and 475 millimicrons wavelength (Fig. 11). Bright f i e l d microscopic examination of fixed, frozen sections of tunic shows that the pigment i s concentrated in the exterior h a l f of the tunic, p a r t i c u l a r l y at the periphery of the tunic. The pigment i s d i f f u s e in the tunic and i s not associated with either granules or c e l l s . The tunic matrix f i b e r s in the v i c i n i t y of the epidermis appear to be c o l o r l e s s . (d) Elemental Analyses: The per cent carbon, hydrogen, oxygen, nitrogen, and s u l f u r i n the tunic and tu n i c i n are compared with these elements in c e l l u l o s e i n Table 19. The per cent hydrogen in t u n i c i n i s about h a l f that i n ce l l u l o s e , 78 FIGURE 11. The absorption spectra of methanol extracts of blood c e l l s and tunic of H. aurantium. .140 .130 .120 co 3 .1101 > co .1001 LU Q .0901 < o £ .0801 o .070 .060 ABSORPTION S P E C T R A OF METHANOL EXTRACTIONS OF BLOOD C E L L S AND TUNIC .050' 400 420 440 460 480 500 W A V E L E N G T H IN MILLIMICRONS 79 TABLE 1 9 . The composition of c e l l u l o s e , tunic, and t u n i c i n in terms of per cent of carbon, hydrogen, oxygen, nitrogen, and s u l f u r . The per cent values for tunic and t u n i c i n are the per cents of the ash free material. The empirical formula, for the computation of per cent compostion of elements in c e l l u l o s e , i s ( c 6 H i o 0 5 ^ n * Cellulose Tunic Tunicin PER CENT Carbon Hydrogen Oxygen Sulfur Nitrogen 4 4 . 4 7 6 . 2 3 4 9 . 3 3 0 0 4 1 . 7 5 3 .26 4 9 . 3 7 0 . 8 8 4 . 7 5 4 2 . 0 4 3 .23 54 .27 0 .07 0 . 4 0 80 while the oxygen in t u n i c i n i s higher than i n c e l l u l o s e . Even a f t e r the acid and base preparation of tunici n , residual nitrogen and s u l f u r are apparent. (e) Reducing Sugar Content of Tunicin: There i s an increasing y i e l d of reducing sugar from t u n i c i n as a function of duration of hydrolysis in 7 2% H2SO4 at room temperature (Fig. 12). The maximum y i e l d of reducing sugar is 73.1% (n = 3, S.E. = 3.1%) of the dry weight of t u n i c i n a f t e r 11 hours hydrolysis in 72% s u l f u r i c acid. Thin layer chromatography of an aliquot from the neutralized eleven hour hydrolysate displays a single spot which corresponds in position, r e l a t i v e to the solvent front, with D-glucose standards. (e) Uronic Acid Analyses: Tunicin hydrolysates, 9 hours in 7 2% H2SO4, show the presence of uronic acids when assayed by the carbazole method. At 0.5, 3, and 9 hours of hydrolysis, the y i e l d of uronic acid, as per cent dry weight, i s 25.8% (n = 3, S.D. = 0.4%), 22.9% (n = 3, S.D. = 0.5%), and 24.7% (n = 3, S.D. = 0.4%). Thin layer chromatography of the neutralized hydrolysates, however, does not reveal spots which correspond to either D-glucuronic or D-galacturonic acid standards. The only spots on these chromatographs correspond to D-glucose i n t h e i r e l u t i o n patterns. (f) Hexosamine Analyses: Hexosamine y i e l d i s expressed as per cent of hydrolyzed weight of tunic. Hydrolyzed weight i s calculated for sample as the weight difference of tunic before and a f t e r h y d r o l y s i s . T r i p l i c a t e samples of tunic, hydrolyzed for 2, 4, 6 and 8 hours in 6N HCl, produce 9.7% (S.E. = 0,7%), 9.3% (S.E. = 0.3%), 11.0% (S.E. = 1.7%), and FIGURE 12. The y i e l d of reducing sugar from t u n i c i n as a function of time of hydrolysis in 72% H 9S0 4. I I 1 I I I 1 3 5 7 9 11 HOURS OF H Y D R O L Y S I S IN 7 2 % H2S0 4 82 8.7% (S.E. = 0.4%) of t h e i r hydroxysed weight as hexosamine a f t e r decontamination of the samples on columns of Dowex r e s i n . The optimal time of hydrolysis i s four hours for maximum y i e l d with l e a s t v a r i a t i o n . S e r i a l elution, with 0.33N HCl through a 48 cm. x 0.8 cm. column of Dowex resin, of pooled 4 hour tunic hydrolysates displays two peaks of Elson-Morgan reactive material. E l u t i o n of a standard, consisting of 2-amino-D-glucose and 2-amino-D-galactose, or e l u t i o n of t h i s standard mixed with hydrolysate, also produces two peaks of Elson-Morgan reactive material. The r e l a t i v e e l u t i o n volumes of the two peaks coincide i n a l l three systems (Fig.13). This indicates that both glucosamine and galactosamine are present in tunic hydrolysates. The r a t i o of glucosamine and galactosamine i s 4.06:1.00. (g) /Amino Acid Analyses: Seventeen amino acids, plus glucosamine, are recoverable from hydrolysis of tunic in 6N HCl for 24 hours. The y i e l d of amino acids and glucosamine, disregarding ammonia, equals 31.8% of the dry weight of tunic. Pronase digestion for 48 hours p r i o r to tunic hydrolysis r e s u l t s in a decrease of y i e l d to 11.9% of the dry weight of tunic, i . e . approximately 65.8% of the recoverable amino acids are susceptible to pronase digestion. The molar r a t i o s of twelve of the amino acids, including glucosamine, in the hydrolysates before pronase digestion d i f f e r s i g n i f i c a n t l y from t h e i r r a t i o s a f t e r pronase digestion (Table 20). A l l of the amino acids are not equally l a b i l e to pronase. L a b i l i t y to pronase i s calculated for each amino acid as the difference in y i e l d per milligram of tunic before and a f t e r enzyme treatment 83 FIGURE 13. The e l u t i o n patterns of glucosamine and galactosamine from a 48 cm. column of Dowex 50W X-8 cation exchange r e s i n . The eluant was 0.33 N HCl. The three systems represent the hydrolysate of tunic, hydrolysate of tunic mixed with glucosamine and galactosamine standards, and a galacto-samine and glucosamine standard. The el u t i o n volumes are r e l a t i v e to the elution volume of glucosamine. 500 450 400 L U 350 < co o X LU JZ 300 250 200 CO < cr o o cr 2 150 100 50 A-•o Standard -© Standard-J-Unknown -A Unknown / \ / J I \ / A V \ / •90 -95 M)0 1-05 1-10 1-15 1-20 1-25 ELUTION VOLUME RELAT IVE TO GLUCOSAMINE 84 divided by the y i e l d before such digestion. Glucosamine and serine are l e a s t susceptible to pronase, and tyrosine and methionine most susceptible (Table 20). The a c i d i c amino acids constitute 22.1% and the basics 11.8% of the amino acids present in the hydrolysate. The basic amino acids do not change s i g n i f i c a n t l y in t h e i r molar concentration upon enzyme treatment whereas the a c i d i c s do. Proline i s present at a concentration of 5.1% before enzyme treatment, and approximately 5.8% a f t e r pronase digestion. 85 TABLE 20. The molar r a t i o s (as per cent) of the eighteen amino acids i n tunic hydrolysates before and after 48 hours incubation with pronase. The per cent of each amino acid which i s pronase l a b i l e i s presented also. The per cent of amino acid l a b i l e to pronase i s the difference in the y i e l d of that amino acid per milligram of tunic before and af t e r pronase divided by the y i e l d of the amino acid before pronase treatment. p represents the l e v e l of si g n i f i c a n c e in the difference of molar r a t i o s before and af t e r enzyme treatment. MOLAR PER CENT PER CENT Before After Pronase Pronase Pronase P L a b i l e AMINO ACID Glucosamine 3.81 7.22 0.001 28.1 Serine 6.74 7.83 0.01 56.1 Proline 5.12 5.75 0.01 57.5 Alanine 5.50 6.23 0.01 57.5 Glutamic 9.00 9.74 0.01 59.1 Lysine 5.79 6.16 N.S. 59.6 Valine 5.55 5.73 N.S. 60.7 Glycine 10.26 10.41 N.S. 62.0 H i s t i d i n e 1.25 1.23 N.S. 63.1 Threonine 5.67 5.43 N.S. 63.8 Argenine 4.73 4.35 N.S. 64.9 Isoleucine 3.31 3.06 0.05 65.1 Leucine 4.37 4.02 0.02 65.3 Phenylalanine 3.70 •3.36 0.05 65.8 Aspartic 13.07 11.68 0.001 66.4 Cysteine 6. 50 4.92 0.001 71.4 Tyrosine 3.70 2.05 0.001 78.6 Methionine 1.85 0.76 0.001 84.6 86 DISCUSSION The morphology of blood c e l l s from ascidians have been investigated by numerous authors (Seeliger and Hartmeyer, 1911; Ohuye, 1936; George, 1939; Peres, 1943; Endean, 1955a and 1960; Andrew, 1962). The blood c e l l s of the ascidians f a l l into three morphological categories; vacuolar c e l l s , amoebocytes, and lymphocytes or stem c e l l s . The f i r s t category includes the vanadocytes (Webb, 1939; Endean, I960), ferrocytes, and t h e i r precursors (Endean, 1955a and 1960). The second category includes the phagocytes. The t h i r d category includes those undifferentiated c e l l s which can divide to form s i m i l a r daughter c e l l s (Peres, 1943 and 1947), d i f f e r e n t i a t e into one of the f i r s t two categories (George, 1939; Endean, 1955a and 1960), or i n i t i a t e budding i n some c o l o n i a l species (Freeman, 1964). The signet ring, compartment c e l l , immature morula, which i s morphologically s i m i l a r to the ferrocyte (Endean, 1955a), and mature morula c e l l s of H. aurantium belong to the f i r s t category. The hyaline amoebocyte, granular amoebocyte, and dispersed v e s i c u l a r c e l l would belong to the second category. The stem, giant stem, and stem with a c i d o p h i l i c vacuoles or granules of Halocynthia belong to the t h i r d category. Quantitative information concerning the concentration of c e l l s per cubic millimeter of blood as either absolute concentration or d i f f e r e n t i a l d i s t r i b u t i o n of c e l l types i s l i m i t e d . Comparative discussion i s d i f f i c u l t because of the d i s p a r i t i e s in typing of blood c e l l s . The name of the c e l l types, used by the author cite d , w i l l be followed by the designation of the supposed analogue i n Halocynthia aurantium in parentheses. Freeman (1964) reported a mean c e l l concentration of 134,000 c e l l s per cubic millimeter of blood i n Perophora  v i r i d i s . Of these, 0.5% were lymphocytes (stem), 5.0% phagocytes (H.A.), 5.0% granular amoebocytes (G.A. and D.V.C.), and 60% green c e l l s (I.M. and M.M.) (Freeman, 1964). The blood of a singl e Ciona i n t e s t i n a l i s contained 43.3% ves i c u l a r and phagocytic c e l l s (Signet and H.A.), 19.2% a c i d o p h i l i c .granulocytes (G.A. and D.V.C.), and 17.1% hyaline amoebocytes (Millar, 1953). In Ph a l l u s i a mammillata, there are 68,000 c e l l s per cubic millimeter of blood; 43% are vanadocytes (M.M. and I.M.), 44% compartment c e l l s , and 1% phagocytes (H.A.) (Endean, 1960). Pyura s t o l o n i f e r a shows a mean c e l l concentration of 37,000 c e l l s per cubic millimeter of blood (n = 12), 70% of these are ferrocytes (M.M. and I.M.), 3% macrophages (H.A.), and 5% lymphocytes (stem) (Endean, 1955a). George (1939) investigated the blood elements of 26 species of tunicates and categorized c e l l s into 8 types. Although he did not present quantitative data, he noted that of hi s 8 c e l l types lymphocytes (stem), macrophages (H.A.), compartment, and signet ri n g c e l l s were present i n a l l 26 species. Ohuye (1936) investigated 3 Japanese species and c l a s s i f i e d the c e l l s into 10 types which c l o s e l y resemble George's (1939) categories. He noted the presence or absence of these categories i n the three species he investigated. There have been numerous reports i n the l i t e r a t u r e of 88 d i f f e r e n c e s i n r e l a t i v e blood c e l l d i s t r i b u t i o n within and between animals as a function of p o s i t i o n within the animal and growth of new structures. Peres (1948a) noted changes i n the r e l a t i v e concentration of d i f f e r e n t blood c e l l types i n the blood spaces of young blastozooids and stolons of Clav e l i n a lepadiformes as a function of blastozooid formation and regression. He noted fluctuations i n the number of Tropfenzellen (signet), c e l l s with metachromatic granules (G.A. and D.V.), and phagocytes (H.A.) as a function of stage of development. In Ciona, the r e l a t i v e numbers of blood c e l l types in subepidermal blood spaces appear to change as a function of time a f t e r detunication (Peres, 1948b). There also appears to be a difference i n c e l l d i s t r i b u t i o n as a function of p o s i t i o n i n the body of Ciona (Millar, 1953), and t h i s i s reported also for other species (Cuenot, c i t e d i n Andrews, 1962). A r e l a t i v e change i n c e l l types as a function of season (George, 1926) and as a function of stolon develop-ment (Freeman, 1964) has been reported i n Perophora v i r i d i s . I t should be noted that both Cla v e l i n a and Perophora are c o l o n i a l animals which reproduce asexually by budding as well as by sexual methods. Freeman (1964) has shown that lymphocytes (stem) are involved in bud formation in Perophora. Comparisons of these data are d i f f i c u l t because of the d i v e r s i t y of methods for gathering blood and counting r e l a t i v e numbers of c e l l s . In P h a l l u s i a (Endean, 1960) and Ciona (Millar, 1953) blood was drawn by cardiac puncture, in Pyura (Endean, 1955a) by excavation of the base of the tunic, and in Ciona 89 and Cla v e l i n a (Peres,1948a and 1948b) c e l l s were counted _in s i t u i n stolons and subepidermal blood spaces a f t e r f i x a t i o n and sectioning of the material. Generally, the numerical data have been based on a r e l a t i v e l y small sample s i z e ; _e.jj. Peres (1948a), in h i s work with Clavelina, used a maximum number of 3 animals for any given time of count. Even though the great v a r i a b i l i t y i n blood c e l l d i s t r i b u t i o n within the blood of a single species has been acknowledged (Seeliger and Hartmeyer, 1911; Endean,1955a), there have been no s t a t i s t i c a l analyses of the concentration of blood c e l l s per cubic m i l l i -meter of blood and t h e i r d i f f e r e n t i a l d i s t r i b u t i o n as a function of weight of animal for large numbers of given species. Halocynthia aurantium, u t i l i z e d f o r blood c e l l counts, were c o l l e c t e d i n the summer and f a l l , 1966. Blood for c e l l counts was drawn from the ventral sinus. The mean number of blood c e l l s per cubic millimeter of blood in Halocynthia, calculated from a sample >of 35 animals, i s lower than that reported for other species. The great v a r i a b i l i t y of c e l l concentration i s evidenced by the large standard error of the mean. Analysis of variance of the number of t o t a l c e l l s per cubic millimeter of blood versus the wet weight of animal shows that the concentration of c e l l s i n the blood does not change as a function of weight of animal. The d i f f e r e n t i a l d i s t r i b u t i o n of the stem c e l l , the stem with a c i d o p h i l i c vacuoles or granules, dispersed v e s i c u l a r c e l l and mature morula c e l l decrease with increased weight of animal. The hyaline amoebocyte increases with increased weight of animal, whereas 90 the granular amoebocyte shows no differences i n r e l a t i o n to weight. The immature morula, compartment and signet c e l l , considered c o l l e c t i v e l y , show a decrease i n r e l a t i v e number with increased weight of animal, although considered as single types none show a change with weight of animal. Since the t o t a l c e l l concentration per uni t blood volume does not show a change with difference in weight of animal, the differences i n r e l a t i v e d i s t r i b u t i o n of p a r t i c u l a r c e l l types r e f l e c t s a r e a l d ifference i n the concentration of p a r t i c u l a r c e l l types per u n i t blood volume. I t should be noted that the r e l a t i v e l y higher number of stem c e l l s i n smaller animals i s i n agreement with the findings of Peres (1947). In general, the mature and immature morula of H. aurantium represents 30% of the blood c e l l s present. This figure i s somewhat lower than reported values for comparable c e l l s in other species (Endean, 1955a arid 1960; Freeman, 1964). The signet r i n g c e l l i n Halocynthia i s at a r e l a t i v e l y low concentration, as i t i s in P h a l l u s i a (Endean, 1960) and Pyura (Endean, 1955a). However, the r e l a t i v e concentration of signet rings, as Tropfenzellen or v e s i c u l a r c e l l s , i s quite high in Cla v e l i n a (Peres, 1948a) and Ciona (Millar, 1953) . In r e l a t i o n to this, signet analogues, the Tropfenzellen (Ries, 1937) and the c e l l with a single refringent grain (Peres, 1948a and 1948b), are reported to be involved in the tunic of Cla v e l i n a and Ciona, whereas the mature morula and i t s analogues i s involved in the tunics of Halocynthia, Pyura (Endean, 1955b) and P h a l l u s i a (Endean, 1961). The object of d e t a i l e d study of the blood elements i n 91 ascidians i s to gain insight into the histogenesis and function of p a r t i c u l a r blood c e l l types. The function of some of the blood c e l l s can be ascertained by d i r e c t investigation of that function, for example phagocytosis. The function of other c e l l types, however, does not lend i t s e l f to d i r e c t i n v e s t i g a t i o n . Consequently, the function of these c e l l types must be inferred from knowledge of phenomena .such as s i t e s of aggregation of blood c e l l s other than in blood, chemical contents of the blood c e l l s in r e l a t i o n to these s i t e s of aggregation, and reaction of blood c e l l s to experimental manipulation of these s i t e s of aggregation. The presence of c e l l s in the tunic of ascidians has been known for sometime (Herdman, 1899; Seeliger and Hartmeyer, 1911; Henze, 1913; Hecht, 1918; and Herdman, 1924). I t has been noted by B e r r i l l (1950) that in compound ascidians p a r t i c u l a r l y those that exhi b i t zooid degeneration, v i r t u a l l y a l l of the blood c e l l types are found in the tunic. There are f i v e c e l l types in the tunic of Herdmania p a l l i d a : large and small e o s i n o p h i l i c c e l l s , a large granular c e l l (D.V.) concentrated at the tunic surface, spherical vacuolar c e l l s and amoeboid c e l l s (Das, 1936). In Cynthia p a p i l l o s a , four c e l l types are found in the tunic: a pigment c e l l which i s concentrated in the basal layers of the tunic, a v e s i c u l a r c e l l , amoeboid c e l l s , and spindle-shaped c e l l s (Seeliger and Hartmeyer, 1911). In t h i s species, St. H i l a i r e (1931) reported three c e l l types in the tunic; a coarsely granular c e l l (M.M.) which is in high concentrations in basal tunic layers 92 and i n the t i p s of growing stolons, a f i n e l y granular c e l l (D.V.) dispersed throughout the tunic and aggregated below areas of t h i n c u t i c l e , and an agranular c e l l (H.A.). In growing stolon, the coarsely granular (M.M.) shows de t e r i o r a t i o n of structure as a function of distance from the epidermis to the exterior of the stolon tunic (St. H i l a i r e , 1931). The concentration of c e l l s i n the tunic of Cynthia  p a p i l l o s a i s reported to be 240 c e l l s per square millimeter of tunic. I f the sections used i n these counts were 10 microns i n thickness, t h i s would amount to 24,000 c e l l s per cubic millimeter of tunic (St. H i l a i r e , 1931). In Ciona i n t e s t i n a l i s , the outer layers of tunic show a higher concentration of c e l l s than the inner and there appears to be three c e l l types present; a granular amoebocyte (G.A. and D.V. C.)., a ve s i c u l a r c e l l (signet), and a phagocyte (H.A.) (Millar, 1953). In Cla v e l i n a lepadiformes, a c e l l with compressed inclusions migrates into the tunic and great numbers are present in stolon tunic. In t h i s species Tropfenzellen (signet) show high r a t i o s of tunic to blood concentration for both young and old stolon; 30/1 for young material and 21/1 for older material. However, in budding chambers, where c u t i c l e formation i s intense, the r a t i o i s 2/1 (Peres, 1948a). Tunic c e l l s i n some species may be derived from pseudohaemoblasts which have migrated into the tunic (Peres, 1947). In Pyura s t o l o n i f e r a , the concentration of c e l l s i n the tunic has been estimated at 33,200 per cubic millimeter of tunic, many of which are ferrocytes (M.M.) (Endean, 1955b). Both blue and green c e l l s (M.M.) have been 93 reported in the tunic of As c i d i a atra (Hecht, 1918). Pigment c e l l s , phagocytes (H.A.), bladder c e l l s , and vanadocytes (M.M.) have been reported in the tunic of Ph a l l u s i a mammillata. Bladder c e l l s are believed to be deteriorated vanadocytes (M.M.) and are concentrated in the outer 3/4 of the tunic (Endean, 1960). Vanadocytes (M.M.) also are present i n the tunic of As c i d i a  pygmaea (Kalk, 1963). The r e l a t i v e numbers of ve s i c u l a r and other c e l l types do not d i f f e r between the body and tunic f l u i d of Chelyosoma siboja (Kobayashi, 1938). In Halocynthia  aurantium, four blood c e l l types are evident in the tunic of body wall and stolon; the mature morula, dispersed v e s i c u l a r c e l l , granular amoebocyte, and hyaline amoebocyte. The t o t a l concentration of c e l l s in the tunic of body wall in H. aurantium i s approximately 28,000 c e l l s per cubic millimeter of tunic. This figure i s of the same order of magnitude as the c e l l u l a r concentration in the tunic of Pyura s t o l o n i f e r a (Endean, 1955b) and Cynthia p a p i l l o s a (St. H i l a i r e , 1931). The p a r t i c u l a r c e l l types in the tunic of H. aurantium are at s i g n i f i c a n t l y higher concentrations than they are in the blood. Further, the concentration of these c e l l types in the tunic of the stolon i s much higher than i t i s in the tunic of the body wall. Both the mature morula and the dispersed v e s i c u l a r c e l l appear to undergo changes in the tunic which preclude t h e i r migration back into c i r c u l a t i o n . The hyaline amoebocyte i s the phagocytic c e l l in the tunic of Halocynthia and apparently has the a b i l i t y to migrate i n and out of the tunic. Both the mature morula and the dispersed v e s i c u l a r c e l l d isplay d e f i n i t e 94 s i t e s of aggregation in the tunic. The mature morula c e l l i s concentrated, almost exclusively, just adjacent to the epidermis, whereas the dispersed v e s i c u l a r c e l l displays i t s most dense concentration at the external periphery of the tunic. This p o s i t i o n i n g of c e l l s in the tunic of Halocynthia agrees e s s e n t i a l l y with the po s i t i o n i n g of analogous c e l l types in Cynthia p a p i l l o s a (St. H i l a i r e , 1931). The large granular c e l l , which aggregates at the periphery of the tunic i n Herdmania (Das, 1936) may correspond to the dispersed v e s i c u l a r c e l l . This p o s i t i o n i n g appears to be reversed i n Ciona which shows i t s greatest concentration of c e l l s in the outer tunic (Millar, 1953) and the bladder c e l l s of Ph a l l u s i a are concentrated in the outer three-quarters of the tunic (Endean, 1960). There appear to be three general categories of c e l l s in the tunic of ascidians; a c i d o p h i l i c vacuolar c e l l s (M.M.), granulated c e l l s (D.V.C. and G.A.), and phagocytic c e l l s (H.A.). In some species there i s evidence of di s c r e t e areas of aggregation for p a r t i c u l a r c e l l types, but the evidence from other species i s incomplete. Growth may be stimulated through injury; but care must be exercised that traumatic phenomena are not confused with growth phenomena. Injury to the tunic of As c i d i a atra results i n an aggregation of blue c e l l s (M.M.) i n the injured area (Hecht, 1918). Seventy-two hours a f t e r injury to the tunic of Phallusia  mammillata, there i s s i g n i f i c a n t tunic regeneration and a large aggregation of c e l l s including vanadocytes (M.M.) and th e i r degradation products (Endean, 1960). Twenty-four to f o r t y - e i g h t hours a f t e r detunication of Ciona, there i s a massive i n f l u x of c e l l s with a s i n g l e r e f r a c t i v e grain (signet) and c e l l s with a c i d o p h i l i c granules and rods (D.V.C.) (Peres, 1948b). Also i n Ciona, during the course of siphon regeneration studies, i t has been reported that 16 hours a f t e r siphon removal, there i s regeneration of the epidermis i n the wound area, that t h i s epidermis i s a c t i v e l y secreting new ground substance for the tunic, and c e l l s with a single or multiple grain (signet) are contributing to ground substance formation (Sutton, 1953) . Afte r 56 hours, most of the c e l l s i n the i n j u r y area are those with many refringent grains (D.V.C.) (Sutton, 1953). In C l a v e l i n a lepadiformes, Tropfenzellen (signet), are reported to be capable of b u i l d i n g new tunic during zooid regression (Ries, 1937). These c e l l s were capable of secreting a t u n i c - l i k e material around foreign objects introduced into the tunic (Ries, 1937). Forty days a f t e r i n j u r y to Cynthia microcosmus, there i s no evidence of new c u t i c l e formation, but in Cynthia p a p i l l o s a , t h i r t y days a f t e r injury, there i s s l i g h t c u t i c l e formation but no spine formation. In both of these cases, however, there i s an aggregation of f i n e l y granular c e l l s (D.V.C.) below the i n j u r y area (St. H i l a i r e , 1931). St. H i l a i r e (1931) does not believe that the f i n e l y granular c e l l s are responsible for c u t i c l e formation since they do not form a d e f i n i t e epithelium. In the i n j u r y areas of Cynthia p a p i l l o s a and Cynthia microcosmus, there i s no resculpturing of tunic layers over the period of observation (St. H i l a i r e , 1931). The e f f e c t of an injury to the tunic of 96 Halocynthia aurantium i s an immediate and s i g n i f i c a n t increase in the concentration of mature morula and dispersed v e s i c u l a r c e l l s in the i n j u r y area. Neither the granular amoebocyte nor the hyaline amoebocytes increase s i g n i f i c a n t l y in the wound area. Even with the increase in mature morula and dispersed v e s i c u l a r c e l l s in the injury area, there is a rapid r e - e s t a b l i s h -ment of the p o s i t i o n a l relationships of the mature morula to the inside and the dispersed v e s i c u l a r c e l l to the outside of the tunic. Injury to the tunic, which includes injury to the epidermis, r e s u l t s in no new tunic formation. In injured tunic, in which the epidermis i s not damaged, there i s a breakdown of mature morula in the area, and the matrix of the tunic i n these areas becomes f i l l e d with mature morula breakdown products as evidenced by coincidence of s t a i n i n g q u a l i t i e s of mature morula contents and the matrix a f t e r injury. This phenomenon also occurs in growing stolons p a r t i c u l a r l y at the t i p of the stolon where the tunic material i s quite t h i n . The dispersed v e s i c u l a r c e l l s r a p i d l y migrate to the edges of the wound and, a f t e r a short time, the presence of a densely staining c u t i c l e material i s evident at the edges of the wound. Injury studies of Halocynthia do not show a s i g n i f i c a n t increase in concentration of hyaline amoebocytes, the phagocyte, over a 15 day period. Both the mature morula and dispersed v e s i c u l a r c e l l are probably immobilized in the tunic. Phagocytosis studies have shown that hyaline amoebocytes can move in and out of the tunic. Consequently, i t can be surmised that the f l u x of hyaline amoebocytes could increase, but that, because 97 of great v a r i a t i o n in s t a t i c content of these c e l l s i n the tunic between animals, t h i s i s e f f e c t i v e l y masked. I t i s apparent that c e r t a i n blood c e l l s are intimately and d i s c r e t e l y involved in the tunic. Further, in Halocynthia i t i s evident that two of these blood c e l l s , mature morula and dispersed v e s i c u l a r c e l l , have s p e c i f i c areas of function in the tunic. The tunic of ascidians can be roughly categorized as either fibrous or membranous (St. H i l a i r e , 1931), and tunic of Halocynthia would f a l l into the fibrous category. Lamination of the tunic of ascidians appears to be widespread, but not of general occurrence (St. H i l a i r e , 1931; M i l l a r , 1953). The family Pyuridae has attained the highest degree of d i f f e r e n t i a t i o n of tunic with both a spinous surface and a fibrous matrix which gives i t an o v e r a l l leathery texture ( B e r r i l l , 1950). However, f i b e r s are not exclusive to the Pyuridae (Herdman, 1899; St. H i l a i r e , 1931). Electron microscope investigations of the tunic of Perophora v i r i d i s reveal that the tunic i s composed of sheets of 50A filaments running p a r a l l e l to the epidermal surface (Deck, et a l . , 1966). Asc i d i a has a gelatinous tunic containing a fibrous matrix (Herdman, 1899). The tunic of Pyura s t o l o n i f e r a i s gelatinous with f i n e f i b e r s i n terlaced in a l l planes, but these f i b e r s are oriented p a r a l l e l to the long axis of the animal i n deeper areas of the tunic next to the body (Endean, 1955b). A condensed dark s t a i n i n g c u t i c l e at the external periphery of the tunic i s of common occurrence. In Cynthia p a p i l l o s a , S t . H i l a i r e (1931) has claimed that the c u t i c l e and spines are a condensation product 98 of the matrix material, and that the granular c e l l s (D.V.C.) do not contribute s i g n i f i c a n t l y to t h e i r chemical composition. In other species Hecht (1918) and Das (1936) have stated that the tunic i s sloughed at the surface with a constant growth from the epidermis. The matrix i s considered, by some authors, to consist of an amorphous ground substance which can be condensed into f i b e r s (St. H i l a i r e , 1931; Godeaux, 1963). St. H i l a i r e (1931) recognized the r e g u l a r i t y of fibrous laminae in the tunic of Cynthia p a p i l l o s a and found, based on a sample of 3 animals, that there was an increase in number of these fibrous laminae with increase i n weight of animal. He maintained that the lamination of the tunic was the r e s u l t of the sea water pressure operating against the tunic material as i t was l a i d down. St. H i l a i r e (1931) further suggested that the r e g u l a r i t y of the f i b r o s i t y was correlated with the c o n t r a c t a b i l i t y of the tunic. The presence of a d e f i n i t e c o r r e l a t i o n between number of laminae and wet weight of animal has been demonstrated in t h i s work for the tunic of Halocynthia  aurantium. The question of the formation of laminae i n Halocynthia i s discussed below. It has been suggested by numerous authors that the blood c e l l s in the tunic are at l e a s t p a r t l y , i f not t o t a l l y , responsible for tunic secretion (Herdman, 1899; Ries, 1937; Endean, 1955a, 1955b, 1960, 1961). Others believe that the blood c e l l s have an organizing, rather than secretory, function in the tunic (St H i l a i r e , 1931; Das, 1936), while s t i l l others maintain that the tunic i s p r i m a r i l y an epidermal secretion 99 (Deck, e_t a l . , 1966). Many of the suggestions for the o r i g i n of tunic depend on histochemical analogy of tunic and blood c e l l s or tunic and epidermis. The majority of f i x a t i o n and histochemical methods, devised for the elucidation of the carbohydrate constituents of tissues, have been developed for vertebrate, and more s p e c i f i c a l l y mammalian, systems. Inter-p r e t a t i o n of histochemical tests for carbohydrates in inverte-brate tissues, p a r t i c u l a r l y some of the older work, may be ambiguous. In order to overcome possible confusion in i n t e r -p r e t a t i o n of histochemical tests used in t h i s work, a b r i e f discussion of t h e i r t h e o r e t i c a l s p e c i f i c i t y and possible sources of a r t i f a c t i s given. For the successful completion of any histochemical reaction, several requirements must be f u l f i l l e d ; the concentration of dye and substrate must be high enough to f a c i l i t a t e v i s u a l i z a t i o n of the product, the substrate must be av a i l a b l e to the dye in both a s t e r i c and chemical sense, and the substrate, and substrate-dye complex, must be n o n - d i f f u s i b l e . The P.A.S. method for the detection of neutral polysaccharides depends on the presence of an a v a i l a b l e v i c i n a l d i g l y c o l of a sugar molecule. The bond between the carbons bearing the hydroxyl groups i s broken by the action of the p e r i o d i c acid with an oxidation of the hydroxyl groups to aldehydes. The S c h i f f ' s reagent i s a c o l o r l e s s fuchsin d e r i v a t i v e which upon reaction with the aldehyde groups forms a red product. The S c h i f f ' s reagent w i l l also react with other aldehyde groups, i f they have not been oxidized further by the periodate. The hydroxyl 100 analogue of the amine group in hexosamine i s also reactive to t h i s method (Hale, 1957; Pearse, 1961; C u l l i n g , 1963). Bauer's reaction i s e s s e n t i a l l y s i m i l a r to the P.A.S. technique except that a d i l u t e chromic acid i s used instead of p e r i o d i c acid (Casselman, 1957). The P.A.S. method appears to he the method of choice because of the s p e c i f i c i t y of reaction of periodate with v i c i n a l d i g l y c o l s r e s u l t i n g in dialdehydes. There are a number of compounds which w i l l give a p o s i t i v e P.A.S. reaction other than carbohydrates, such as phospholipids. Certain cases exhib i t a negative P.A.S. where a p o s i t i v e reaction would be expected; the P.A.S. reaction of c h i t i n decreases with increased s c l e r o t i z a t i o n (Hale, 1957), and keratosulfate spotted on f i l t e r paper gives a negative reaction ( Q u i n t a r e l l i , 1968). P.A.S., as well as mucicarmine and mucihaematin stains, has a greater a f f i n i t y for e p i t h e l i a l mucins than for connective tissue acid mucopolysaccharides (Curran, 1961). The s t a i n i n g of acid mucopolysaccharides generally depends on the i o n i c character of the substrate and dye. Five of the tests used in t h i s work for the detection of acid mucopoly-saccharides depend on the i o n i c i n t e r a c t i o n of an anionic acid group i n the substrate with a c a t i o n i c group i n the dye* C o l l o i d a l iron, methylene blue, azure A at pH 1.5 and 4.0, and t o l u i d i n e blue. The l a s t four dyes are a l l thiazines (Baker, 1958). The pKa's of the s u l f a t e and carboxyl groups found in acid mucopolysaccharides are below pH 7.0. At neutral pH these acid groups w i l l be deprotonated and available to the basic dyes used. The pKa of the carboxyl groups i s approximately 4.0 and that of the s u l f a t e groups about 1.5 (Szirmai, 1963). By using buffered s t a i n i n g solutions, the approximate pH, and hence pKa, at which the dye ceases to be found by the substrate can be determined. This i s the t h e o r e t i c a l p r i n c i p l e under-l y i n g the methylene blue e x t i n c t i o n t e s t (Pearse, 1961) . By s t a i n i n g at a con t r o l l e d pH of 1.5 or 4.0, one can determine whether there i s a predominance of carboxyl or s u l f a t e groups i n the substrate (Spicer, 1960; Szirmai, 1963) . If the substrate acid groups are within a c r i t i c a l distance of each other and are of a high density, then, with the thiazine dyes, the phenomena of spectral s h i f t , metachromasia, may occur (Sylven, 1954). With c l o s e l y spaced acid substrate groups of high density, s t a i n i n g with t o l u i d i n e blue should r e s u l t in a red colored product or gamma metachromasia (Sylven, 1954). Alc i a n blue i s a copper phthalocyanin. The exact mechanism of dye-substrate i n t e r a c t i o n i s unknown, but i t i s supposed that the copper group of the phthalocyanin interacts with the anionic groups of the substrate. L i t t l e i s known of the mechanism of reaction of aldehyde fuchsin with substrates, other than the f a c t that i t appears p r e f e r e n t i a l l y to s t a i n acid mucopoly-saccharides (Halmi, 1953). The s u l f a t e and carboxyl groups of acid mucopolysaccharides are subject to chemical modification. Carboxyl groups can be methylated with a c i d i c methanol, thus blocking the reactive s i t e . The conditions used in acid methylation w i l l desulfate s u l f u r i c acid esters (Spicer, 1960). Methylated carboxyl groups can be saponified, i . e . demethylated by treatment with base (Spicer, 1960). Substrates, which lose 102 t h e i r a f f i n i t y for acid mucopolysaccharide stains upon methylation and do not regain i t upon saponification, may be considered as sulfated. Substrates, which regain t h e i r dye a f f i n i t y a f t e r s a p o n i f i c a t i o n of methylated tissues, may be considered carboxylated (Spicer, 1960). These methods are subject to spurious i n t e r p r e t a t i o n and a r t i f a c t s . The metachromatic dyes of commercial source may contain extraneous red dyes ( Q u i n t a r e l l i , 1968). C o l l o i d a l iron techniques w i l l give a p o s i t i v e reaction to protein, nucleic acids, and collagen ( Q u i n t a r e l l i , 1968; Curran, 1961). Many early workers used lead acetate or Zenker's f i x a t i v e s , which are now considered of l i t t l e use i n preserving acid mucopolysaccharides (Curran, 1961). The use of 1% HCl in methanol for methylation procedures can cause hydrolysis of substrates ( Q u i n t a r e l l i , 1968). The methylene blue e x t i n c t i o n t e s t and the use of buffered solutions of azure A are s p e c i f i c only i f ce r t a i n other tiss u e components can be excluded, such as ribonucleic acids (Pearse, 1961). Therefore, to c o r r e c t l y i n t e r p r e t histochemical tests, one must u t i l i z e a wide range of reactions, include as many controls as possible, and, i d e a l l y , couple histochemical tests with independent chemical and biochemical analyses. With these points in mind, the theories of tunic o r i g i n from either blood c e l l s or epidermis based upon histochemical analogy can be judged c r i t i c a l l y . The tunic of Ciona reacts p o s i t i v e l y to the Bauer's reaction and i s strongly metachromatic, but these reactions are negative for the epidermis. Two c e l l types in the tunic, the 103 c e l l with a c i d o p h i l i c granules and rods (D.V.C.) and the c e l l with a single acidophils vacuole (signet or H.A.) are p o s i t i v e to Bauer's reaction, but show no metachromasia. The t h i r d category of c e l l s i n the tunic, the c e l l with a single refringent grain (signet), displays a weak carbohydrate reaction (Peres, 1948b). Bierbauer and Vagas (1962) report that the tunic of Ciona contains a c i d i c polysaccharides, which are i n highest concentration adjacent to the epidermis and decrease i n s t a i n i n g i n t e n s i t y p e r i p h e r a l l y . The mesenchymal c e l l s in the tunic were negative for acid mucopolysaccharides but strongly p o s i t i v e to the P.A.S. technique (Bierbauer and Vagas, 1962). The tunic of C l a v e l i n a lepadiformes also gives a strong Bauer's reaction and metachromasia, p a r t i c u l a r l y at the l e v e l of the epidermis. The f i b e r s of the tunic s t a i n p o s i t i v e for p r o t e i n (Peres, 1948a). The Tropfenzellen (signet), which migrates into tunic, and i s reported to be capable of secreting tunic, contains a glycoprotein (Ries, 1937). I t i s accepted generally that there i s both protein and carbohydrate present i n the tunic material (St. H i l a i r e , 1931). Recently i t has been reported that there i s l i t t l e to no protein present i n the tunic of Pyura and P h a l l u s i a (Endean, 1955b and 1961). The tunic of Pyura s t o l o n i f e r a histochemically shows the presence of both a neutral and an a c i d i c polysaccharide. The neutral polysaccharide i s r e s i s t a n t to various chemical reagents (Endean, 1955b). In Pyura the compartment c e l l and ferrocyte (M.M.) give p o s i t i v e protein and carbohydrate reactions histochemically (Endean, 1955a). The tunic of P h a l l u s i a mammillata stains 104 p o s i t i v e for neutral and a c i d i c polysaccharides and negative for p r o t e i n . The carbohydrate material i s r e s i s t a n t to hyaluronidase treatment (Endean, 1961). The vanadocyte (M.M.) in t h i s species contains both protein and carbohydrate, and i t i s suggested that i t secretes t u n i c i n (Endean, 1961) . In Perophora v i r i d i s , whose tunic displays a strong p o s i t i v e P.A.S. reaction, the vanadocyte (M.M.) w i l l not take up t r i t i a t e d glucose, although the epidermis w i l l . This l a b e l eventually appears in the tunic (Deck, _et a l . , 1966). St. H i l a i r e (1931) reported that the tunics of Cynthia  p a p i l l o s a , Cynthia microcosmus, and Ph a l l u s i a mammillata contain both protein and c e l l u l o s e . The tunics of Chelyosoma  productum, C o r e l l a willmeriana, A s c i d i a paratropa, B o l t e n i a v i l l o s a , Halocynthia igaboj a, and Pyura haustor s t a i n p o s i t i v e l y with a l c i a n blue, c o l l o i d a l iron, and aldehyde fuchsin. The tunics of these ascidians also display a b r i l l i a n t red metachromasia with t o l u i d i n e blue (Smith, unpublished). Even where there i s an analogy of staining properties between blood c e l l s and tunic material, i t has been maintained that the epidermis i s s t i l l the tunic secreting t i s s u e (Peres, 1948b; Sutton, 1953). In other cases, the function of the mature morula analogue i s i n dispute (Endean, 1955a, 1955b, 1960, and 1961; Deck, et a l . , 1966). The tunic matrix f i b e r s of Halocynthia aurantium s t a i n with those dyes which would indicate that i t i s a sulfated acid mucopolysaccharide. Upon methylation and saponification, 105 h o w e v e r , d i s c r e p a n c i e s a r i s e . T h e e f f e c t o f m e t h y l a t i o n a n d s a p o n i f i c a t i o n u p o n t h e a f f i n i t y o f t h e m a t r i x f i b e r s f o r a z u r e A a t p H 1 . 5 i s a r e t u r n o f s t a i n i n g i n t e n s i t y , w h i c h a l t h o u g h n o t a s h i g h a s u n t r e a t e d t i s s u e s , i s o f a n o r d e r o f m a g n i t u d e g r e a t e r t h a n f o u n d i n m e t h y l a t e d t u n i c . S i n c e c a r b o x y l g r o u p s s h o u l d b e p r o t o n a t e d a t t h i s p H a n d s u l f a t e g r o u p s s h o u l d h a v e b e e n r e m o v e d b y t h e m e t h y l a t i o n t e c h n i q u e s , t h e r e s u r g e n c e o f s t a i n i n g i n t e n s i t y may b e e x p l a i n e d o n t h e b a s i s o f a n i n c o m p l e t e m e t h y l a t i o n , e x c e p t i o n a l l y s t r o n g l y d i s s o c i a t e d c a r b o x y l g r o u p s , o r t h e p r e s e n c e o f a n u n c h a r a c t e r i z e d s u b s t r a t e . O f t h e b l o o d c e l l s w h i c h c o n c e n t r a t e i n t h e t u n i c o f H a l o c y n t h i a a u r a n t i u m , t h e h i s t o c h e m i s t r y o f h y a l i n e a m o e b o c y t e s a n d g r a n u l a r a m o e b o c y t e s i s n o t c o n s i s t e n t w i t h t h a t o f e i t h e r t h e s p i n e s o r m a t r i x f i b e r s . T h e h y a l i n e a m o e b o c y t e i s e s s e n t i a l l y a p h a g o c y t e . T h e g r a n u l a r a m o e b o c y t e i s a n e n i g m a . I t d o e s n o t p o s s e s s s t a i n i n g c h a r a c t e r i s t i c s w h i c h a r e common t o a n y o f t h e t u n i c s t r u c t u r e s . I t d o e s n o t c h a n g e i n r e l a t i v e n u m b e r i n t h e b l o o d w i t h c h a n g e i n w e i g h t . T h i s may b e a c c e p t e d a s a n a r g u m e n t t h a t i t s f u n c t i o n i s r e l a t i v e t o a g i v e n v o l u m e o f b l o o d r a t h e r t h a n a t a r g e t o r g a n o r a r e a . I t i s p o s s i b l e t h a t i t i s a c o a g u l o c y t e , b u t t h e r e i s n o d e f i n i t e e v i d e n c e f o r t h i s . A l t h o u g h l i t t l e c a n b e s a i d a b o u t t h e c o n t r i b u t i o n o f m a t u r e m o r u l a c e l l s t o t h e c h e m i c a l s u b s t a n c e o f t h e t u n i c m a t r i x f r o m h i s t o c h e m i c a l e v i d e n c e , i t s h o u l d b e n o t e d t h a t a 106 change i n a f f i n i t y for p a r t i c u l a r dyes takes place i n the mature morula c e l l before i t i s degraded to the tunic v e s i c u l a r types (Table 14), and that the mature morula c e l l s s t a i n for presence of iron and protein rather than carbohydrate. The dispersed v e s i c u l a r c e l l stains p o s i t i v e for v i c i n a l d i g l y c o l s by the P.A.S. technique, but only i f the tissue i s fixed with cyanuric chloride with N-methyl morpholine. With other f i x a t i v e s the r e s u l t s are anomalous. The substrate in t h i s case may be quite d i f f u s a b l e , but e f f e c t i v e l y sequestered by the cyanuric chloride f i x a t i v e and not other f i x a t i v e s . The v e s i c l e s of the dispersed v e s i c u l a r c e l l s t a i n equivocally for a c i d i c polysaccharides. There i s a d i s s i m i l a r i t y of st a i n i n g properties between tunic dispersed v e s i c u l a r c e l l s and spines, but there i s some s i m i l a r i t y of s t a i n i n g properties between blood dispersed v e s i c u l a r c e l l s and the spines, e.g. azure A, at pH 4.0, aldehyde fuchsin, and mercury bromophenol blue. Further, i f R.N.A. st a i n i n g can be used as an index of synthetic a c t i v i t y , the tunic dispersed v e s i c u l a r c e l l i s in a c t i v e . In Halocynthia aurantium, the histochemical s i m i l a r i t y between the tunic matrix f i b e r s and epidermis, the consistent R.N.A. sta i n i n g properties of the epidermis, the v i s i b l e continuation of tunic f i b e r s with the epidermal c e l l s , and the f a i l u r e of new tunic material to form i n areas of injured epidermis, a l l support the hypothesis that the 107 epidermis i s the major tunic secreting t i s s u e . The data brought forth i n t h i s i n v e s t i g a t i o n do not support the hypothesis that the mature morula c e l l contributes s i g n i f i c a n t l y to the tunic carbohydrate component. The function of the mature morula c e l l i n the tunic may be related to i t s iron content. D i f f e r e n t species of ascidians concentrate d i f f e r e n t t r a n s i t i o n metals (Endean, 1953; C a r l i s l e , 1958; Levine, 1962; Kokubu and Hidaka, 1965). The f i r s t report of metal accumulation was by Henze (1911) for vanadium. Henze (1932) suggested that vanadium accumulation may be re l a t e d to the presence of c e l l u l o s e in ascidian tunics. Endean (1955a, 1955b, I960, and 1961) reported that ferrocytes and vanadocytes (M.M.) contain t u n i c i n precursors and are responsible for t u n i c i n secretion. Evidence from t h i s study does not support the hypothesis i n the case of the mature morula c e l l . In Halocynthia  aurantium, there i s a high concentration of iron i n both the blood c e l l s and the tunic. The iron, i n the blood c e l l s , i s in a non-dialyzable f r a c t i o n , and, a f t e r treatment with hydrogen peroxide and o x a l i c acid, can be histochemically v i s u a l i z e d i n the signet ring, the compartment, immature, and mature morula c e l l s . The s t a i n i n g i n t e n s i t y varies both i n and between c e l l types. I t has been demonstrated that the mature morula c e l l concentrates i n the tunic j u s t above 108 the epidermis. In t h i s region fibrous laminae are being formed from the fibrous extrusions of the epidermal c e l l s . Lamination of the tunic i s a property which Halocynthia aurantium shares with Cynthia p a p i l l o s a (St. H i l a i r e , 1931). St. H i l a i r e (1931) suggested that pressure of sea water caused the lamination of tunic. I t i s questionable whether the pressure of sea water could form the lamination. H. aurantium does not d i s p l a y any gas space, and the i n t e r i o r of the animal i s , for the most part, open to the sea. I t i s d i f f i c u l t to under-stand how a pressure d i f f e r e n t i a l would be formed. At the depth at which Halocynthia i s found, the f l u c t u a t i o n of pressure due to t i d a l changes represents only a f r a c t i o n of the t o t a l pressure. Further, the presence of bands of laminae, diverse i n thickness, can not be explained on the basis of external pressure. The presence of the bands of diverse laminae may be explained on the basis of a seasonal v a r i a t i o n i n reserve substances. Halocynthia has one major spawning period a year (Smith unpublished) from l a t e spring to e a r l y summer. In the l a t e f a l l and early winter, most of the reserve substances probably are u t i l i z e d for gonadal production, thus decreasing the amount avail a b l e for tunic formation. Fluctuation of metabolite l e v e l s as a function of the gonadal cycle has been reported for several marine 109 invertebrates (Barry and Munday, 1959; Nimitz and Giese, 1964). I t i s suggested that the lamination of the tunic i s caused by the mature morula c e l l acting at the l e v e l of the epidermis. There are several facts which support t h i s suggestion. The tunic i s not secreted by the epidermis as a sheet, but as sing l e f i b e r s . In the region where these f i b e r s coalesce into a lamina, the mature morula c e l l s aggregate and breakdown, l o s i n g t h e i r c h a r a c t e r i s t i c s t a i n i n g properties. This may i n d i c a t e either a c a t a l y t i c condensation e f f e c t or contribution of p r o t e i n to the tunic material, or both. At s i t e s of r a p i d l y growing tunic (stolons) there i s a s i g n i f i c a n t l y higher concentration of mature morula c e l l s . At the t i p of the growing stolon, where no d e f i n i t e lamination occurs, the mature morula c e l l s are concentrated at the tunic periphery. The function of the stolon i s to form a hold-fast. Since the mature morula c e l l s are located at the periphery, condensation of f i b e r s would be determined by the contours of the substratum. In the laboratory, where stolons grow with no substrate contact, there i s an increasing r e g u l a r i t y of lamination proceeding from the growing t i p of the stolon to the body w a l l . Further the p o s i t i o n a l r e l a t i o n s h i p of mature morula c e l l s in the laboratory grown stolon, except for the growing t i p , resembles that of the body wall, with the greatest concentration of mature morula c e l l s j u st 1 1 0 e x t e r i o r to the epidermis. The arrangement of f i b e r s in natural stolons i s i r r e g u l a r . Injury to body wall tunic r e s u l t s in a massive migration of mature morula c e l l s to the wound area. In t h i s case, the mature morula c e l l s break down i n i t i a l l y at the periphery of the wound. In these peripheral areas of mature morula breakdown there i s a coalescence and condensation of lamina, and the area i s occluded with mature morula break-down products. Later, there i s a re-establishment of the p o s i t i o n a l r e l a t i o n s h i p for the mature morula as found i n normal tuni c . The i n i t i a l reaction i s probably a traumatic reaction, which i s followed by a stimulated growth e f f e c t . The mature morula c e l l decreases in r e l a t i v e number per cubic millimeter of blood with increased weight of animal. It is concentrated i n the body wall tunic i n a r e s t r i c t e d area adjacent to the epidermis. In thi s p o s i t i o n i t degenerates into a tunic v e s i c u l a r c e l l . This process of d e t e r i o r a t i o n probably excludes the p o s s i b i l i t y of further d i f f e r e n t i a t i o n or e x i t from the tunic. The decrease i n r e l a t i v e concentration of mature morula c e l l s may be a function of t h i s p o s i t i o n a l r e l a t i o n s h i p in the tunic. The concentration of mature morula c e l l s j u s t above the epidermis has the character of a surface accumulation. Since the surface and volume of a sphere or cyl i n d e r do not increase at the same rate, there could be a r e l a t i v e decrease i n mature morula c e l l s in respect to the volume of blood with increased s i z e of animal, while a constant number of mature morula c e l l s per u n i t surface area i s maintained. In a sense t h i s can then be considered a form of, or a d e r i v a t i v e of, allometric growth (Reeve and Huxley, 1 9 4 5 ) . This e f f e c t may be further i n t e n s i f i e d , i f the mature morula c e l l has a primary growth function. The s p e c i f i c growth rate of organisms decreases with increase age and i t i s generally accepted that increase in s i z e r e f l e c t s increase in age (Medawar, 1 9 4 5 ) . With increased s i z e of H. aurantium, the r e l a t i v e decrease of mature morula c e l l s would be expected, i f these c e l l s are involved i n growth and are measured i n r e l a t i o n to some s i z e phenomena such as units of blood volume. The same arguments, put forward to explain the decrease in r e l a t i v e number of mature morula c e l l s as a function of increased s i z e of ascidian, are v a l i d for the dispersed v e s i c u l a r c e l l . I t would be expected that those c e l l types which are precursors to the mature morula and dispersed v e s i c u l a r c e l l also would decrease in r e l a t i v e number with increased s i z e of animal as do the stem, the stem with a c i d o p h i l i c vacuoles or granules, and the vacuolar types. The dispersed v e s i c u l a r c e l l appears also to have a d i s c r e t e function i n the tunic. I t aggregates at the exterior edge of the tunic, i t increases, and maintains, a s i g n i f i c a n t l y higher number of c e l l s i n injured tunic, i t concentrates at i t s highest l e v e l s i n the r a p i d l y growing tunic of stolons, i t maintains a p o s i t i o n a l r e l a t i o n s h i p i n the stolons, i n body wall tunic i t i s c l o s e l y associated with the spines, and i t s v e s i c l e s appear to be composed of protein material. In natural stolons, laboratory grown stolons, body wall tunic, and injured tunic, two properties stand out; a high concentration of dispersed v e s i c u l a r c e l l s at the periphery of the tunic material and the presence of a c u t i c l e or spines i n these areas. The spines are of a uniform s i z e and r e g u l a r l y d i s t r i b u t e d over the surface of body wall tunic i r r e s p e c t i v e of weight of animal. As the ascidian grows, there must be an increase i n tunic surface area and, concomitant with t h i s increase, there must be an absolute increase i n the number of spines. For the e f f e c t i v e growth of new spines, the most obvious region for the i n i t i a t i o n of t h i s growth i s from the i n t e r p a p i l l a r y surface between older spines. These regions are p a r t i c u l a r l y r i c h i n dispersed v e s i c u l a r c e l l s . In laboratory grown stolons, which have no substrate contact, there i s a graded development of spines over the length of the stolon from the growing t i p , which has no spines, to the juncture of the stolon with the body wall, where f u l l y developed spines are evident. C u t i c l e material, but not spines, i s evident, at the growing t i p s of natural stolons and laboratory grown stolons, at the edges of wounds, and along the length of natural stolons. At the growing t i p s of stolons and, i n i t i a l l y , at the edges of wounds, there i s no p o s i t i o n a l separation of mature morula and dispersed v e s i c u l a r c e l l s . The c u t i c l e and spines have analogous s t a i n i n g properties. I t has been suggested that c u t i c l e and spines of Cynthia p a p i l l o s a are composed of the same substance as the tunic matrix, but that the mode of deposition d i f f e r s (St. Hilaire, 1931) . In Halocynthia aurantium, the histochemical reactions of the spines are c l e a r l y d i f f e r e n t from those of the matrix. The dispersed v e s i c u l a r c e l l at the edge of tunic material displays 113 c h a r a c t e r i s t i c s which could indicate that i t f u n c t i o n a l l y i s expended. The v e s i c l e s are smaller and more d i f f i c u l t to stain, the cytoplasm has l o s t i t s a b i l i t y to take up stain, the dispersed v e s i c u l a r c e l l i n p o s i t i o n never stains for the presence of R.N.A., and the nucleus appears necrotic. I t i s proposed that the dispersed v e s i c u l a r c e l l has the function of forming c u t i c l e and spines. The spines w i l l be elaborated upon completion of i n i t i a l growth processes, which may involve the mature morula c e l l , and i f the environmental conditions allow a free surface and s u f f i c i e n t time for growth. In natural stolon, there i s a surface to surface contact, which may i n h i b i t the elaboration of spines. In injury repair studies, the edge of the wound mimics the t i p s of growing stolons in that both mature morula and dispersed v e s i c u l a r c e l l s are present and not p o s i t i o n a l l y separated i n i t i a l l y . Given s u f f i c i e n t time, the edge of the wound might elaborate spines. An analogous s i t u a t i o n would be the laboratory grown stolon. At the growing t i p no spines are present, but there i s a graded development of spines along the length of stolon with f u l l y developed spines at the juncture of body wall and stolon. In a l l cases, includ-ing the surface of i n t e r p a p i l l a r y areas of body wall tunic, a c u t i c l e i s secreted. Subsequently, the c u t i c l e may be elaborated into spines or not depending on environmental factors. The pigmentation of ascidians has been investigated by several authors. Tsuchiya and Suzuki (1952) isol a t e d several pigments from the tunic of Cynthia r o r e t z i . The major pigments present were alcohol soluble cynthiaxanthin and astaxanthin. 1 1 4 These compounds also have been isol a t e d from Halocynthia  p a p i l l o s a (Lederer, c i t e d in Fox, 1953) . Methanol soluble p i g -ments, which have coincident absorption maxima, are extractable from the tunic and blood c e l l s of H. aurantium. Frozen sections of H. aurantium tunic show that the pigment i s concentrated p r i m a r i l y in the outer l i m i t s of the tunic, and that the basal layers of the tunic, near the epidermis, are c o l o r l e s s . The pigment of the tunic i s d i f f u s e and not granular. Injured tunic and growing stolon d i s p l a y a very weak pigmentation. Although both the dispersed v e s i c u l a r c e l l and the pigmentation occur in the exterior h a l f of the tunic, the pigmentation areas are much wider than the dispersed v e s i c u l a r c e l l aggregation area. Because of the weak pigmentation of stolon and injured tunic which are high in number of dispersed v e s i c u l a r c e l l s , and since examination of l i v e dispersed v e s i c u l a r c e l l s does not d i s c l o s e any yellow or orange pigmentation, the dispersed v e s i c u l a r c e l l has not been considered a pigment c e l l . The hyaline amoebocyte has a phagocytic function. Examination of l i v e hyaline amoebocytes shows that they occasionally contain a large yellow concretion or granule. These c e l l s may have a secondary function of carrying pigment to the tunic. The r e l a t i v e number of hyaline amoebocytes increases with increased weight of animal. One possible explanation of t h i s phenomenon i s related to the h a l f - l i v e s of various c e l l types. Both the dispersed v e s i c u l a r and mature morula c e l l s apparently deteriorate in the tunic material. There i s no obvious area or organ in Halocynthia aurantium 115 which displays large aggregations of d e t e r i o r a t i n g hyaline amoebocytes. The function of the dispersed v e s i c u l a r and mature morula c e l l s probably depends on t h e i r d e t e r i o r a t i o n . The function of the hyaline amoebocyte, as a phagocyte, could suggest continuing v i t a l i t y . I f the h a l f - l i f e of hyaline amoebocytes exceeds that of the dispersed v e s i c u l a r and mature morula c e l l s , there might be an accumulation of these c e l l s with time, assuming that t h e i r rate of d i f f e r e n t i a t i o n does not decrease with age of animal. The stem c e l l type i n Halocynthia agrees morphologically with the lymphocyte type of other authors. It i s generally believed that the lymphocyte gives r i s e to the other blood c e l l types in the ascidians (George, 1939; Peres, 1943; Endean, 1955a, and 1960). This inves t i g a t i o n does not o f f e r any evidence which would negate these hypotheses. I t is suggested, however, that the development of other c e l l types passes through the stem with a c i d o p h i l i c vacuoles or granules stage. Consequent to t h i s the granular amoebocyte, hyaline amoebocyte, and dispersed v e s i c u l a r c e l l s develop. The histochemical analyses of the tunic of Halocynthia  aurantium displayed c h a r a c t e r i s t i c s which would indicate that i t i s an acid mucopolysaccharide. There was also evidence for the presence of protein. Early reports suggested that c e l l u l o s e should be detectable i n tunic of ascidians (Seeliger and Hartmeyer, 1911), but the P.A.S. technique resulted in a negative reaction for the tunic under a v a r i e t y of conditions. I t was decided, therefore, to analyze the tunic both chemically 116 and b i o c h e m i c a l l y to attempt to c l a r i f y the chemical composition o f t u n i c i n t h i s s p e c i e s . The water c o n t e n t o f f r e s h t u n i c o f a s c i d i a n s v a r i e s ; i n C y n t h i a p a p i l l o s a 6 5% o f the f r e s h weight o f t u n i c i s water (St. H i l a i r e , 1931), i n Pyura s t o l o n i f e r a 94.8% (Endean, 1955b), i n P h a l l u s i a mammillata 94.1% (Endean, 1961), i n A s c i d i a mentula 87% (Henze, 1913), and i n H a l o c y n t h i a aurantium 81%. H y d r o l y t i c treatment of d r y t u n i c w i t h a c i d or base r e s u l t s i n a l o s s o f 50% o f the d r y weight o f t u n i c i n H a l o c y n t h i a  aurantium. Upon s i m i l a r treatment , the t u n i c o f P_. mammillata l o s e s 42% o f i t s d r y weight (Abderhalden and Zemplen, 1911) , and t h e r e i s a 50% l o s s i n the cases o f A s c i d i a mentula and A s c i d i a  mammillaris ( W i n t e r s t e i n , 1894). The a c i d and base treatment o f t u n i c i s designed to supply a r e l a t i v e l y n i t r o g e n f r e e p r o d u c t ; i t i s , i n e f f e c t , a d e - i o n i z i n g and d e - p r o t e i n i z i n g p r o c e d u r e . The p r o d u c t o f such treatment, however, does not prove g e n e r a l l y to be completely f r e e o f n i t r o g e n . In P h a l l u s i a  mammillata such treatment f u r n i s h e s a p r o d u c t which i s s t i l l 0.05% n i t r o g e n ( K r a s s i g , 1954), i n A s c i d i a mentula and A. mammillaris t h e r e i s a r e s i d u a l o f 0.13% n i t r o g e n ( W i n t e r s t e i n , 1894). In H a l o c y n t h i a aurantium t u n i c i n , d e r i v e d by acid/base treatment o f t u n i c , d i s p l a y s 0.40% n i t r o g e n which i s a p p r o x i m a t e l y 8.5% o f the o r i g i n a l n i t r o g e n i n d r y t u n i c . T u n i c i n from H a l o c y n t h i a has a r e s i d u a l s u l f u r content o f 0.07% which r e p r e s e n t s approximately 8% o f the o r i g i n a l s u l f u r p r e s e n t i n the t u n i c . The elemental composition o f t u n i c i n o f v a r i o u s s p e c i e s 117 has been investigated for carbon, hydrogen, and oxygen. P h a l l u s i a mammillata t u n i c i n is 44.76% carbon and 6.45% hydrogen (Krassig, 1954). Other authors (cited in Seeliger and Hartmeyer, 1911) report carbon compositions ranging from 43.40% to 45.38%, and hydrogen compositions ranging from 5.68% to 6.47% i n t u n i c i n of P h a l l u s i a mammillata. The elemental composition of t u n i c i n from Cynthia p a p i l l o s a i s reported as 43.20% or 44.6% carbon and 6.16% or 6.1% hydrogen (cited in Seeliger and Hartmeyer, 1911). Pyura s t o l o n i f e r a t u n i c i n has a carbon content of 40.7% and hydrogen content of 6.2% (Endean, 1955b). Halocynthia t u n i c i n has a much lower hydrogen content, 3.23% and higher oxygen content, 54.27%, than any of these reports. The carbon content of H. aurantium t u n i c i n , 42.04%, i s higher than the value reported by Endean (1955b) for Pyura, but lower than values reported for other species (see above). This lower hydrogen content, when considered with evidence given below, may indicate that the composition of t u n i c i n i s something other than c e l l u l o s e . One of the c e l l u l o s i c properties of tunici n , which was reported at an early date, i s i t s d i s p e r s a b i l i t y in Schweitzer's reagent (cited i n Seeliger and Hartmeyer, 1911). Schweitzer's reagent (cupric ammonium hydroxide), concentrated s u l f u r i c acid, 85% phosphoric acid, and other solutions of strong ions disperse c e l l u l o s e f i b e r s by disrupting c r y s t a l l i n e forces, such as hydrogen bonds and van der Waal's forces, of micelles formed between adjacent l i n e a r polymers of c e l l u l o s e (Cowling, 1963; Jermyn and Lang, 1963). A s i m i l a r procedure, u t i l i z i n g hot 118 concentrated l i t h i u m thiocyanate, has been used to disperse c h i t i n (Gottschalk, 1966). There are c o n f l i c t i n g reports of the d i s p e r s a b i l i t y of tunic and t u n i c i n . Schweitzer's reagent w i l l not disperse the tunics of P h a l l u s i a mammillata and Cynthia p a p i l l o s a , but i t w i l l swell them to a degree (St. H i l a r i e , 1931). The addition of cold concentrated s u l f u r i c acid to the tunic of P_. mammillata w i l l cause i t s rapid d i s s o l u t i o n , but the f i b e r s i n the tunic of Cynthia p a p i l l o s a w i l l not dissolve u n t i l the system i s heated (St. H i l a i r e , 1931). Later reports state that t u n i c i n from P_. mammillata w i l l disperse (Krassig, 1954) or w i l l not disperse (Pruvot-Fol, 1951) i n Schweitzer's reagent. The acid hydrolysis product of the tunic of Pyura s t o l o n i f e r a i s not dispersable by Schweitzer's reagent (Endean, 1955b). Tunicin from Halocynthia does not disperse in Schweitzer's reagent over a 48 hour period nor in hot concentrated l i t h i u m thiocyanate. Tunicin from Halocynthia i s charred by cold concentrated s u l f u r i c acid, and i t w i l l swell, but not disperse, in 85% phosphoric acid. The f a i l u r e of most of these reagents to have an e f f e c t on Halocynthia tunic could be due to a number of factors; there may be an extraordinary degree of c r y s t a l l i n i t y in the t u n i c i n f i b e r s , c r o s s - l i n k i n g of l i n e a r carbohydrate polymers by linkages other than the beta 1,4, or the presence of n o n - c e l l u l o s i c moieties which contribute to the f i b r i l l a r structure. The l i t e r a t u r e indicates that t u n i c i n i s composed p r i m a r i l y of glucose, and various studies have isol a t e d both glucose and glucose d e r i v a t i v e s . Abderhalden and Zemplen (1911) 119 obtained the octacetylated d e r i v a t i v e of ce l l o b i o s e from t u n i c i n of P h a l l u s i a mammillata. The y i e l d of acetylated d e r i v a t i v e from P h a l l u s i a was 18% of the weight of tunicin, which was less than could be obtained from f i l t e r paper by the same procedure. E a r l i e r , glucose was i d e n t i f i e d as the major constituent of t u n i c i n hydrolysates from Ascidia mentula and A s c i d i a mammillaris based on o p t i c a l rotatory properties (Winterstein, 1894). Zechmeister and Toth (1934) i s o l a t e d c e l l o t r i o s e , c e l l o t e t r o s e , and cellohexose from t u n i c i n of P h a l l u s i a mammillaris. They further reported obtaining approximately 98% of the weight of t u n i c i n as glucose as determined by copper reduction, iodine number, and o p t i c a l r o t a t i o n . Tsuchiya and Suzuki (1952) obtained a series of oligosaccharides from ce l l o b i o s e to cellopentose which were i d e n t i f i e d by paper chromotography, from the tunic of Cynthia  r o r e t z i . I t i s apparent that the basic constituent sugar of t u n i c i n i s glucose. Halocynthia t u n i c i n w i l l y i e l d approximately 75% of i t s dry weight as glucose, i d e n t i f i e d by th i n layer chromatography, a f t e r 11 hours hydrolysis in 72% s u l f u r i c acid. No doubt some degradation r e s u l t s from the acid treatment (Neuberger and Marshall, 1966),. and the glucose content of the t u n i c i n i s probably higher than 75%. Unfortunately, strong acid hydrolysis of t u n i c i n does not reveal the types of g l y c o s i d i c bonds present. Such treatment w i l l cleave a v a r i e t y of g l y c o s i d i c bonds. The hydrolysis does demonstrate unequivocally that the major constituent of Halocynthia t u n i c i n i s glucose. 120 Because t u n i c i n gives a negative P.A.S. reaction, but has a f a i n t p o s i t i v e a l c i a n blue a f f i n i t y , consideration was given to the p o s s i b i l i t y that the carbohydrate residue might contain uronic acids. Aliquots of a 72% s u l f u r i c acid hydrolysis, samples over an 9 hour period, yielded consistant values of approximately 25% of the dry weight of t u n i c i n as uronic acid. The addition of 3.8 parts of water to the reaction mixture for uronates resulted in a c o l o r l e s s s o l u t i o n . This procedure has been u t i l i z e d for the i d e n t i f i c a t i o n of uronic acids i n hydrolysates (Dische, 1947). Thin layer chromotography of the neutralized hydrolysates does not reveal the presence of either glucuronic or galacturonic acid. Uronates are very d i f f i c u l t to i s o l a t e since they are destroyed by hydrolysis (Gardell, 1961). Hyaluronidase treatment of t u n i c i n does not y i e l d either reducing sugars of uronates and there i s no change in f i b r i l l a r structure of t u n i c i n with t h i s treatment. The presence of uronates i s , therefore, doubted as a constitutent of t u n i c i n . The a l c i a n blue s t a i n i n g of t u n i c i n may be due to non-specific binding of the dye, and the c o l o r i m e t r i c determination of uronates in the acid hydrolysis may be spurious due to glucose contamination ( B i t t e r and Muir, 1962) . Other properties of t u n i c i n have been reported which indicate i t s s i m i l a r i t y to c e l l u l o s e of plants. The i n t r i n i s i c v i s c o s i t y of t u n i c i n derived from P_. mammillata is s i m i l a r to that of plant c e l l u l o s e (Krassig, 1954). Electron d i f f r a c t i o n patterns of A s c i d i e l l a aspersa are comparable to those of plant c e l l u l o s e (Hall and Saxl, 1960). X-ray d i f f r a c t i o n patterns 121 of P h a l l u s i a nigra tunic resemble those of plant c e l l u l o s e (Spence and Richards, 1940), but such i d e n t i f i c a t i o n may not be r e l i a b l e , p a r t i c u l a r l y i f n o n - c e l l u l o s i c material i s present (Round, 1965). The ultimate question of the composition of t u n i c i n depends on the d e f i n i t i o n of c e l l u l o s e . Generally, c e l l u l o s e i s considered to be a l i n e a r unbranched polymer of glucose units g l y c o s i d i c a l l y linked by beta 1,4 linkages. The presence of cross l i n k s has been suggested, but not substantiated. I f cross l i n k s are present, t h e i r contribution to t o t a l linkages would be small. I t i s d i f f i c u l t , in any case, to demonstrate that c e l l u l o s e i s a completely uniform polymer (Whitaker, 1963). The degree of polymerization of c e l l u l o s e can range from 15 to 10,000 (Cowling, 1963) and the range that has been obtained for the t u n i c i n of P h a l l u s i a mammillata i s well within these figures, 700 to 3,500 (Krassig, 1954). I t should be remembered that the t u n i c i n of P h a l l u s i a has not always been reported to be dispersable by Schweitzer's reagent (St. H i l a i r e , 1931) as i t was by Krassig (1954) . Most of these studies have been concerned with t u n i c i n which has been derived from tunic by acid and base treatment. There have been few attempts to ascertain the chemical composition of tunic before such treatments. Dried tunic i s approximately 50% non-carbohydrate material (see above). The presence of c h i t i n i n the tunic has been suggested (Pruvot-Fol, 1951) , but, generally, i t i s considered that no c h i t i n i s present i n the ascidians (Sharon, 1965) . I t i s s u r p r i s i n g that, with the number of reports of metachromatic reaction of tunic and l a t e r of acid mucopolysaccharides in tunic, few authors have investigated the tunic for the presence of hexo-samines. I t i s not s u r p r i s i n g that e a r l i e r workers did not f i n d hexosamine in tu n i c i n , because the a l k a l i treatment, u t i l i z e d in i t s preparation, can deaminate hexosamine (Neuberger, Marshall, Gottschalk, 1966). Even with t h i s treatment there i s generally r e s i d ual nitrogen in t u n i c i n . Endean (1955b and 1961) reported the presence of Elson-Morgan reactive material in the tunic of Pyura s t o l o n i f e r a and P h a l l u s i a mammillata. The Elson-Morgan reaction i s susceptible to interference from amino acids and other materials (Boas, 1953; Gardell, 1955) unless decontamination methods are employed. Endean (1955a) did, however, i d e n t i f y hexosamine from the blood c e l l s of Pyura by ionophoresis. Acid hydrolysates of tunic from Halocynthia, a f t e r decontamination on Dowex cation exchange r e s i n columns, d i s p l a y approximately 5% of the dry weight of tunic as hexosamine. The hexosamines were i d e n t i f i e d as glucosamine and galactosamine, which are present in a r a t i o of approximately 4/1. Whether the hexosamine i s d i r e c t l y linked to the carbo-hydrate of the tunic or is a constituent of protein of tunic i s not known, but the resistance of hexosamine to pronase digestion would indicate that i t i s close to, i f not d i r e c t l y involved with, the carbohydrate f r a c t i o n (see below). Although most authors agree that there i s considerable p r o t e i n in the tunic of ascidians (St. H i l a i r e , 1931; Peres, 1948a; H a l l and Saxl, 1961; Godeaux, 1963), some do not hold 12 3 t h i s view (Endean, 1955b and 1961). The histochemical tests of the tuni c of Halocynthia indicated that protein was present. The protein f r a c t i o n of the tunic was investigated by acid hydrolysis and chromotography of amino acids before and a f t e r pronase digestion of tunic. Pronase i s a p r o t e o l y t i c enzyme of wide s p e c i f i c i t y , possessing the c a p a b i l i t y of breaking peptide bonds which are the s p e c i f i c substrates of at l e a s t ten other common p r o t e o l y t i c enzymes (Nomoto _et a l . , 1960) . Although the tunic amino acid content, before and a f t e r pronase digestion, was analyzed for molar r a t i o s to ascertain s i g n i f i c a n t differences due to pronase treatment, i t was found that the y i e l d of amino acids between samples, expressed as micromoles per gram dry weight of tunic, showed very small v a r i a t i o n within the before and a f t e r pronase treatment groups. As a consequence, the y i e l d of amino acids before and a f t e r pronase digestion were sui t a b l e f o r the determination of the l a b i l i t y of p a r t i c u l a r amino acids to pronase treatment. If i t i s assumed that the closer to the carbohydrate a p a r t i c u l a r amino acid i s located the less susceptible i t w i l l be to pronase digestion because of s t e r i c hindrances, then amino acid analyses before and a f t e r p r o t e o l y t i c digestion w i l l indicate the amino acids which are, at least, in close proximity to the carbohydrate. Seventeen amino acids and glucosamine were recoverable from acid hydrolysates of tunic from Halocynthia  aurantium. Four amino acids and glucosamine showed s i g n i f i c a n t increased i n molar r a t i o s a f t e r pronase digestion. The y i e l d of glucosamine was considerably less than that obtained from 4 124 hours hyd r o l y s i s ; but t h i s would be expected considering the acid l a b i l i t y of hexosamines. Serine, p r o l i n e , alanine, and glutamic acid a l l have higher molar r a t i o s a f t e r pronase digestion, which, when considered in terms of pronase l a b i l i t y , would indicate that they are l e a s t susceptible to p r o t e o l y t i c digestion. Serine i s acid l a b i l e under hydrolysis conditions (Blackburn, 1968) and as much as 10% can be l o s t during a 24 hour hydrolysis period at 110° C. (Eastoe, 1966). Tryptophan i s p a r t i c u l a r l y unstable to acid hydrolysis conditions and both glutamine and asparagine w i l l be broken down by acid conditions to asparate, glutamate, and ammonia (Eastoe, 1966). Both asparate and glutamate are acid stable, however (Eastoe, 1966; Blackburn, 1968). The s u l f u r containing amino acids are also acid l a b i l e , but t h e i r preservation i s f a c i l i t a t e d by anaerobic conditions (Eastoe, 1966). There are f i v e probable sources of ammonia i n the acid hydrolysates of tunic; asparagine, glutamine, serine, threonine, and glucosamine. The ammonia production i n acid hydrolyses generally can be attributed to asparagine and glutamine (Blackburn, 1968), but in p r o t e i n -polysaccharide complexes the cource of ammonia can be much more complex. The ammonia produced by acid hydrolysis of tunic i s 52.5% l a b i l e to pronase digestion, but the v a r i a t i o n in y i e l d between samples was greater for ammonia than for any of the amino acids. The v a r i a b i l i t y of ammonia standards was also quite high. Consequently, l i t t l e i n t e r p r e t a t i o n of ammonia y i e l d can be furnished based on t h i s system. Several amino acids are reported to form the l i n k between carbohydrate and protein i n glycosaminoglycans and glyco-125 p r o t e i n s ; i n egg albumin, the l i n k i s through the one p o s i t i o n of the sugar i n aspartyl glycosylamine (Neuberger et _al., 1966) , i n chondroitin-4-sulfate, the hydroxyl group of serine i s g l y c o s i d i c a l l y linked to the carbohydrate (Muir, 1968), threonine as well as serine may be involved i n c h i t i n protein linkages (Gottschalk, 1966) as well as in s k e l e t a l k e r a t i n s u l f a t e (Roden, 1968), and serine has been indicated as the l i n k i n heparin (Roden, 1968). The p o s s i b i l i t y of ester l i n k -ages through the terminal carboxyl groups of glutamic and aspartic acid cannot be discounted (Neuberger et a l . , 1966; Roden, 1968). The s u s c e p t i b i l i t y of glucosamine, serine, and glutamic acid to p r o t e o l y t i c digestion i s low in Halocynthia tu n i c . Considered in the l i g h t of evidence from other systems, the p o s s i b i l i t y that serine and glucosamine are involved i n the p r o t e i n carbohydrate linkage i n the tunic of Halocynthia i s quite high. The weight loss of tunic upon acid and base treatment i s approximately 50% of i t s dry weight. Pronase treatment of tunic r e s u l t s in a weight loss of approximately 30%. I f the 50% weight loss due to acid and base can be considered as protein, then pronase hydrolysis s p l i t s o f f approximately 60% of the p r o t e i n present. Post-pronase h y d r o l y t i c y i e l d of amino acids account for 12% of the dry weight of tunic. Therefore, the recovery of amino acids a f t e r acid hydrolysis i s 60% e f f i c i e n t . I f the serine recovery a f t e r pronase treatment i s extrapolated to 100% from 60%, and i f i t i s assumed that a l l serine i s involved in protein-carbohydrate l i n k s and that the carbo-126 hydrate component i s e s s e n t i a l l y a glucose polymer, there would be approximately one serine per thirty-seven glucose units of the carbohydrate component. The c a l c u l a t i o n of hexosamine involvement i n the protein polysaccharide l i n k i s more d i f f i c u l t because of the greater l a b i l i t y of hexosamine to acid h y d r o l y s i s . I f the average y i e l d of hexosamine obtained upon 4 hours hydrolysis of tunic represents hexosamine involved i n linkages; then approximately 10% of the weight of t u n i c i n i s hexosamine and there would be approximately one hexosamine per ten glucose molecules in the carbohydrate. If the hexosamine y i e l d from post-pronase amino acid analysis of tunic i s used in the c a l c u l a t i o n of hexosamine i n carbohydrate-protein linkage estimates, then there would be approximately one hexosamine unit per 27 glucose units i n the carbohydrate. These calu c l a t i o n s assume, among other things, that the recovered serine and hexosamine are involved d i r e c t l y i n the carbohydrate-protein linkages and that the hexosamine i s not a part of the in t e g r a l carbohydrate polymer but forms the l i n k between carbohydrate polymer and pro t e i n . The calc u l a t i o n s do not account for the degradation through acid hydrolysis of serine and hexosamine, and i n t h i s respect can be taken as minimum values. I t should be kept i n mind, that these are approximate values whose si g n i f i c a n c e relates to the kind and order of magnitude of possible carbohydrate-protein linkages in the tunic of Halocynthia aurantium. Proline i s among the amino acids l e a s t susceptible to pronase digestion i n Halocynthia tunic. No hydroxyproline was found i n 1 2 7 the hydrolysates of tunic either before of a f t e r pronase digestion. After collagenase and elastase treatment of A s c i d i e l l a aspersa tunic, there is a protein remnant associated with the polysaccharide material, which i s 50-60% p r o l i n e and glycine, 2% hydroxyproline, and 8% v a l i n e . This residue was higher in b a s i c amino acids, 11% and lower i n a c i d i c amine acids, 9%, than e l a s t i n (Hall and Saxl, 1961) . Collagenase treatment r e s u l t s in an 80% decrease in hydroxyproline and elastase treatment i n a 40% decrease of valine, from values p r i o r to enzyme treatment i n t h i s animal (Hall and Saxl, 1961). I t i s believed that the protein has the properties of both collagen and e l a s t i n (Hall and Saxl, 1961). Collagenase has a f a i r l y s p e c i f i c a c t i v i t y , breaking p r i m a r i l y glycine-proline bonds, but elastase i s a protease with a wide s p e c i f i c i t y towards neutral amino acids (White, Smith, Handler, 1964). Inverte-brate collagens are reported to have 25.5 to 32.3% glycine (Eastoe, 1968), but the glycine content of Halocynthia tunic, even a f t e r pronase digestion, i s equal to about a t h i r d of t h i s . In fact, the glycine content of pronase treated tunic i s lower than that found i n protein complexes of chondroitin-4-sulfate, chondroitin-6-sulfate, or dermatan s u l f a t e a f t e r extensive p r o t e o l y t i c treatment (Hoffman, 1968). Proline content of Halocynthia tunic, before or a f t e r pronase, i s lower than that found i n e l a s t i n , r e s i l i n , or collagen, but i t exceeds the l e v e l s found i n f i b r o i n (Rudall, 1968). The glycine content (31.8%) of f i b r o i n , however, i s much higher than that of tunic. I t would appear that the protein of Halocynthia tunic does not show an 128 amino acid composition which i s related to either collagen or e l a s t i n . However, t h i s does not exclude the p o s s i b i l i t y of these types of molecules, since amino acid analyses without knowledge of the amino acid sequence does not define the type of protein. The tunic of Halocynthia aurantium displays h i s t o -chemically the properties of an a c i d i c polysaccharide with the presence of hexosamines. The carbohydrate portion of the tunic appears to consist e s s e n t i a l l y of glucose. Biochemical substrates can often be characterized by t h e i r s u s c e p t i b i l i t y to s p e c i f i c enzymes. Tunic and t u n i c i n were treated with hyaluronidase, chitinase, and c e l l u l o s e to ascertain the s u s c e p t i b i l i t y of these materials to enzymatic degradation. Chitinase does not attack tunic, pronase treated tunic, or t u n i c i n . The lack of e f f e c t of t h i s enzyme, as measure by released reducing sugar and Elson-Morgon reactive material, would indicate that a l i n e a r polymer of N-acetyl-D-glucosamine, as found in c h i t i n , i s not present, or i f present, i s not accessible to chitinase action. Hyaluronidase incubation of tunic, pronase treated tunic, and t u n i c i n does not release either reducing sugar or hyaluronate. Subsequent sectioning and s t a i n i n g of hyaluronidase treated tunic demonstrates, that although a l c i a n blue s e n s i t i v i t y i s decreased, there i s no increase i n P.A.S. s e n s i t i v i t y of the t i s s u e . T e s t i c u l a r hyaluronidase was chosen because of i t s r e l a t i v e l y wide s p e c i f i c i t y towards acid mucopolysaccharide substrates (Walker, 1961) . Even so, there i s no disordering 129 of f i b r i l l a r structure of the tunic as has been reported in the case of Pyura s t o l o n i f e r a (Endean, 1955b). Ce l l u l a s e from commercial sources i s a mixture of several enzymes including beta-D-(1-3)-glucanase, amylase, xylanase, polygalacturonase, alpha-glucosidase, beta-glucosidase, and other enzymes (Reese and Mandels, 1963). However, the major component (Cx) hydrolyzes c e l l u l o s e to small, low molecular weight fragments which are further degraded by various beta-glucosidases in association with Cx (Bernfeld, 1962) . One would expect, upon incubation of c e l l u l o s e with c e l l u l a s e , a rapid preliminary breakdown of c e l l u l o s e followed by a progressive decrease in sugar release due to r e f r a c t o r y portions of the c e l l u l o s e polymer ( H a l l i w e l l , 1961) which are probably the highly structured c r y s t a l l i n e micelle portions of the micro-f i b r i l s . The degree of c r y s t a l l i n i t y of the m i c r o f i b r i l s can be decreased by swelling the c e l l u l o s e in 85% phosphoric acid (Cowling, 1963). Walseth has obtained 80% s o l u b i l i z a t i o n of cotton f i b e r s a f t e r 6 days incubation i n c e l l u l a s e following phosphoric acid swelling (cited i n H a l l i w e l l , 1963). With phosphoric acid swollen c e l l u l o s e powder, 56% s o l u b i l i z a t i o n has been attained i n 22 hours of incubation with c e l l u l a s e . With an excess of enzyme, 90% s o l u b i l i z a t i o n in t h i s system has been achieved in the same period of time (Halliwell, 1961). Halocynthia tunic i s not susceptible to c e l l u l a s e degradation, and a f t e r pronase treatment i t i s not susceptible to c e l l u l a s e unless pretreated with phosphoric acid. Phosphoric acid swollen pronase treated tunic displays a release of reducing sugar, 130 upon c e l l u l a s e incubation, which decreases with time. Renewal of the c e l l u l a s e incubation medium, to insure that enzyme denaturation was not causing the decrease in rate of degradation, and reswelling with phosphoric acid does not restore the o r i g i n a l rate of reducing sugar release. This i s interpreted to s i g n i f y an increasing u n a v a i l a b i l i t y of substrate, which could be due to a number of factors; p a r t i c u l a r l y r e f r a c t o r y c r y s t a l l i n e structure of the carbohydrate, covalent c r o s s - l i n k s between carbohydrate polymers, or interference from r e s i d u a l p r o t e i n . Approximately 20% of the dry weight of pronase treated tunic is released by c e l l u l a s e action as reducing sugar. Pronase treatment causes a 30% loss in weight of tunic, and acid and base treatment causes approximately a 50% loss in weight. Consequently, the 20% y i e l d of pronase tunic would equal approximately 28% of the weight of t u n i c i n . I f the protein-polysaccharide l i n k i n tunic i s spaced along the carbohydrate polymer at distances of 10 to 37 glucose units (see above), then i t i s conceivable that areas of the carbo-hydrate between l i n k s would be av a i l a b l e for c e l l u l a s e action and that approximately 28% of the sugar could be released. Tunicin i s much more susceptible to c e l l u l a s e digestion. Incubation with c e l l u l a s e r e s u l t s i n an immediate release of reducing sugar. This i n i t i a l release of reducing sugar can be increased by about 30% by p r i o r treatment of the t u n i c i n with phosphoric acid. The maximum y i e l d of reducing sugar, obtained from phosphoric acid swollen t u n i c i n , i s approximately 55% of the o r i g i n a l weight of t u n i c i n a f t e r 14 days incubation. 131 Deproteinization and th i n layer chromotography of the 14 day c e l l u l a s e supernatant reveals one d e f i n i t e spot which corresponds to glucose. The presence of small amounts of ce l l o b i o s e cannot be discounted even though i t was not detectable i n th i n layer chromotograms. The rate of release of reducing sugar from phosphoric acid swollen tunic cannot be re-established by subsequent reswelling and renewal of c e l l u l a s e media. This i s interpreted to indicate that af t e r i n i t i a l degradation there are fewer available s i t e s for c e l l u l a s e action. Tunicin material is prepared by an acid and base treatment which should remove the protein. As a re s u l t , i f the protein-polysaccharide l i n k s have been hydrolyzed by t h i s treatment, one would expect a greater y i e l d of reducing sugar from t u n i c i n unless, other factors interfered. I t seems u n l i k e l y that c r y s t a l l i n e carbohydrate structures would survive three successive phosphoric acid swelling treatments. The major components of c e l l u l a s e are s p e c i f i c for a beta-1, 4-glucosyl-glucan. The resistance of t u n i c i n to c e l l u l a s e digestion may indicate that bonds other than the beta-1,4 are present. Therefore, the carbohydrate component of tunic must be considered chemically more complex than c e l l u l o s e . Further, a major proportion of the tunic is protein which appears to be chemically linked to the carbohydrate component. Consequently, the tunic matrix should be regarded as a protein-polysaccharide complex rather than homogeneous carbohydrate. 132 SUMMARY 1. Ten morphological blood c e l l types are recognized in Halocynthia aurantium. The mature morula, dispersed v e s i c u l a r c e l l , stem c e l l , and stem c e l l with a c i d o p h i l i c vacuoles or granules decrease in d i f f e r e n t i a l d i s t r i b u t i o n with increased weight of animal. The hyaline amoebocyte increases in d i f f e r e n t i a l d i s t r i b u t i o n with increased weight of animal. 2. There i s no s i g n i f i c a n t difference i n blood c e l l concentration per cubic millimeter of blood as a function of weight of animal. 3. The mature morula, dispersed v e s i c u l a r c e l l and hyaline amoebocyte, are at s i g n i f i c a n t l y higher concentrations in the tunic of the body wall and stolon than they are in the blood. The mature morula i s concentrated in the tunic just peripheral to the epidermis and the dispersed v e s i c u l a r c e l l i s concentrated at the external l i m i t s of the tunic. The hyaline amoebocyte i s a phagocyte. 4. Upon injury to the tunic, the mature morula and dispersed v e s i c u l a r c e l l s increase s i g n i f i c a n t l y i n the wound area over a 15 day period. The p o s i t i o n a l relationships of these c e l l s are ra p i d l y reinstated in the tunic. 5. The tunic consists of three components: the epidermis, the matrix, and c u t i c u l a r spines. The matrix consists of fibrous laminae which increase in number with increased weight of animal. 6. Halocynthia concentrates both iron and titanium. The 133 highest concentration of iron, r e l a t i v e to dry weight, i s i n the blood c e l l s and tunic. Histochemically iron i s demonstrable in the mature morula, immature morula, compartment, and signet c e l l s . 7. Methanol extracts of the blood c e l l s and tunic d i s p l a y coincident absorption spectra. 8. Histochemistry of tunic reveals that the matrix resembles acid mucopolysaccharide with protein material present. The epidermis stains i n an analogous manner to the matrix. The spines do not display histochemical r e l a t i o n s h i p to either matrix or epidermis. The blood c e l l s do not stain for the presence of polysaccharide material. 9. The s t a i n i n g analogy of the signet ring, compartment c e l l , immature morula, and mature morula, indicate a re l a t i o n s h i p of these c e l l types. The signet ring, compartment and immature morula, considered c o l l e c t i v e l y , also show a decrease in d i f f e r e n t i a l d i s t r i b u t i o n with increased weight of animal. The staining analogy of the blood c e l l types coupled with the changes i n d i s t r i b u t i o n with weight of animal indicate the histogenetic pathways for the d i f f e r e n t i a t i o n of blood c e l l s . 10. Based on p o s i t i o n a l relationships and concentration i n the tunic, injury repair studies, and the morphology of stolon and body wall tunic, the functions of the mature morula and dispersed v e s i c u l a r c e l l i n the tunic are suggested. 11. Fresh tunic i s approximately 80% water. Dry tunic i s approximately h a l f protein and h a l f carbohydrate. 134 12. The carbohydrate component of the tunic i s r e s i s t a n t to a number of dispersing agents, i s negative to P.A.S. technique, i s not susceptible to chitinase nor hyaluronidase, w i l l release 55% of i t s dry weight as glucose af t e r 14 days c e l l u l a s e incubation, w i l l release approximately 7 5% of i t s dry weight as glucose upon acid hydrolysis, and displays an elemental composition which i s lower i n hydrogen and higher in. oxygen than c e l l u l o s e . 13. Approximately 5% of the dry weight of tunic i s hexosamine; both glucosamine and galactosamine are present in a r a t i o of 4 to 1. 14. 60% of the tunic protein i s susceptible to pronase digestion. The tunic protein has an amino acid composition which does not resemble either collagen, e l a s t i n , or other common s t r u c t u r a l proteins. Amino acid analyses of tunic before and a f t e r pronase treatment reveals that p a r t i c u l a r amino acids, such as serine and glucosamine, are more r e s i s t a n t to pronase than others. This i s interpreted to indicate t h e i r involvement in the pr o t e i n -polysaccharide linkage. 15. The histochemical, biochemical, and chemical data brought forth i n t h i s study are inconsistant with the hypothesis that the tunic consists p r i m a r i l y of c e l l u l o s e . These data indicate that the carbohydrate component of tunic i s more complex than c e l l u l o s e , and that the protein contribution to tunic structure i s s i g n i f i c a n t . 135 LITERATURE CITED Abderhalden, E., and G. Zemplen. 1911. P a r t i e l l e Hydrolyse der Tunicatencellulose. Bildung von Cellobiose. Hoppe-Seyler's Z. f. ph y s i o l . Chem. 72:58-62. Andrew, W. 1961. 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