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Nature and function of the ecsoma in Tubulovesicula lindbergi (hemiuridae: platyhelminthes) D’Silva, Joseph 1977

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THE NATURE AMD FUNCTION OF THE ECSOMA IN TUBULOVESICULA LINDBERGI (HEMIURIDAE: PLATYHELMINTHES) by JOSEPH ^ LVSILVA M.Sc, The U n i v e r s i t y of Dacca, 1970 A THESIS.SUBMITTED ;IN PARTIAL FULFILLMENT OF THE- REQUIREMENTS FOR THE DEGREE OF MASTER OF- SCIENCE i n THE DEPARTMENT OF ZOOLOGY We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA May, 1977 f^ cT) Joseph D'Silva, 1977. In presenting th is thes is in p a r t i a l fu l f i lment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f ree ly ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of th is thes is for scho la r ly purposes may be granted by the Head of my Department or by his representat ives . It is understood that copying or pub l ica t ion of th is thes is for f inanc ia l gain sha l l not be allowed without my wri t ten permission. Department of The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date / ( i ) ABSTRACT Some hemiurid parasites have a body proper, the soma, and a tail-appendage, the ecsoma, which i s protractable out of, and r e t r a c t a b l e ^ i n t o , the soma. The nature and functions of the ecsoma. have not been investigated to date, and the hemiurid Tubulovesicula  l i n d b e r g i was used to.determine these. Since the par a s i t e l i v e s i n the stomach of f i s h , pH and s a l i n i t y may a f f e c t the p r o t r a c t i o n and r e t r a c t i o n of the ecsoma. It was assumed that the soma and the ecsoma are not s i m i l a r structures, and the tegument covering the two segments would be d i f f e r e n t . I t was presumed that amino acid could be transported across the tegument of the ecsoma, and thus the ecsoma could function as an organ of absorption. H i s t o l o g i c a l and histochemical methods -were employed to determine the differences between soma and ecsoma. Ecsomas were l i g a t e d , and whole worms were incubated i n a medium containing r a d i o a c t i v e glycine to determine i f glycine could be transported across the tegument. Tests were conducted to detect the secretion of p r o t e o l y t i c enzymes by the ecsoma. The r e s u l t s showed pH and s a l i n i t y had no e f f e c t on the p r o t r a c t i o n of the ecsoma. The somal tegument was a b i - l a y e r and the ecsomal tegument a t r i - l a y e r . Acid phosphatase a c t i v i t y was found on the ecsomal tegument only. The arrangement of the muscle layers beneath the ecsomal tegument was the reverse of that i n the soma. Three types of I ( i i ) cells were found in the parenchyma in the soma and ecsoma. Parenchymal tissues occupied the hulk of the space in the ecsoma. The tegument of the ecsoma accumulated glycine against a concentration gradient. It secreted proteolytic enzymes. The results above indicated that the tegument on the soma and the ecsoma was, indeed-, different. The tegument of the ecsoma was metabolically active, capable of absorbing glycine and secreting proteolytic enzymes. Therefore, the possibility of the ecsoma functioning in the absorption of amino acids from the surrounding media i s discussed. ( i i i ) TABLE OF CONTENTS Page ABSTRACT i TABLE OF CONTENTS i i i LIST OF TABLES v LIST OF FIGURES AND ILLUSTRATIONS v i ACKNOWLEDGMENTS v i i i I. INTRODUCTION 1 II . REVIEW OF LITERATURE a) The soma and the ecsoma 4 b) C r i t i c i s m of various theories 8 c) The tegument 12 I I I . MATERIALS AND METHODS a) Observations i n the f i e l d 17 b) Location and posture of flukes 18 c) Observations i n the a; laboratory 19 d) E f f e c t of pH on the ecsoma 19 e) E f f e c t of s a l i n i t y on the ecsoma 20 f) P r o t r a c t i o n of the ecsoma 20 g) Morphological, h i s t o l o g i c a l studies 21 h) Enzyme histochemistry 23 i ) Uptake of C 1 4 glycine 24 j) Tests for p r o t e o l y t i c enzymes 26 ( i v ) Page IV. RESULTS a) Observations i n the f i e l d 28 b) Observations i n the laboratory 29 c) E f f e c t of pH on the ecsoma 30 d) E f f e c t of s a l i n i t y on the ecsoma 31 e) Morphological and histoehemieal studies 31 i ) The tegument 31 i i ) Musculature 33 i i i ) Parenchyma 34 ,f) Enzyme histochemistry 36 g) Uptake of C1^ glycine 37 h) Tests f o r p r o t e o l y t i c enzymes ^ 37 V. DISCUSSION • 39 VI. SUMMARY 47 VII. TABLES 48 VIII. FIGURES AND ILLUSTRATIONS 55 IXI LITERATURE CITED 66 (v) LIST OF TABLES Page TABLE Ia. Incidence of i n f e s t a t i o n and incidence of i n f e c t i o n of f l a t f i s h by Tubulovesicula  l i n d b e r g i hQ TABLE l i b . Summary of T_^_ l i n d b e r g i i n f e c t i o n s 49 TABLE I l a . Variations i n pH i n f l a t f i s h stomach and condition of ecsoma 51 TABLE lib.. P o s i t i o n of T;_ l i n d b e r g i i n stomach of f i s h examined i n the f i e l d 52 TABLE l i e . Orientation of T. l i n d b e r g i mouth in.the stomach of f i s h examined i n the f i e l d 52 TABLE l i d . P o s i t i o n of T\_ l i n d b e r g i i n stomach of f i s h examined i n the laboratory 52 TABLE l i e . Orientation of T. tliindbergi mouth i n the-stomach of f i s h examined i n the laboratory 52 TABLE I I I . E f f e c t of pH on the ecsoma of T_^_ l i n d b e r g i 53 TABLE TV. E f f e c t of s a l i n i t y on the p r o t r a c t i o n of the ecsoma and the s u r v i v a l of T. l i n d b e r g i 54 (vi) LIST OF FIGURES'AND ILLUSTRATIONS Figure Page 1 ; Cystophorous e e r c a r i a 5.5 2 Tegument of F a s c i o l a hepatiea - electron, microscope study .56 3 Tubulovesi'cula l i n d b e r g i 57 h Tolerance of T\_ l i n d b e r g i to s a l i n i t y 58 5 " B a l l o o n - l i k e " i n f l a t i o n s on ecsomal tegument i n whole mount 59 6 Extra-tegumental coat on ecsoma i n whole mount 59 7 Somal tegument meeting ecsomal tegument 59 8 Tegument of the soma 60 9 Tegument of the ecsoma 60 10 " B a l l o o n - l i k e " i n f l a t i o n s on ecsomal tegument i n l o n g i t u d i n a l sections 60 11 LongitudinalseSection. Ecsoma showing l o n g i t u d i n a l and c i r c u l a r muscles 6l 12 Transverse Section. Ecsoma with r e t r a c t o r muscles 6l 13 ' L.S. Retractor muscles i n soma and ecsoma 6l lU T.S. Retractor muscles'around caecum 6l 15 Type 1 c e l l 62 16 Type 2 c e l l 62 17 Type 3 cells 62 18 L.S. A c i d phosphatase on ecsoma-»tegument 63 L.'S. Acid phosphatase l o c a l i z e d on surface of tegument ' 63 I ( v i i ) Figure • Page 20 Whole mount. Ecsoma with acid phosphatase 63 21 Graph. Glycine uptake by ecsoma •- 6k 22 L y t i c a c t i v i t y of ecsomal homogenate 65 23 23 L y t i c a c t i v i t y around ecsoma 65 (vii'i) ACKNOWLEDGMENTS I would l i k e to acknowledge here a debt that I owe to several people. I am mostly indebted to .Dr. J . R. Adams who suggested the problem for research and allowed me to carry on the present work i n h i s laboratory under his counselling. The members of my research committee, Dr. P. Ford, Dr. W. S. Hoar, Dr. J . P h i l l i p s are also to be thanked for t h e i r advice. I thank Miss Daphne Hards for sharing her expertise on microtechniques with me. Thanks are also due to Dr. Hild a Ching who gave me a patient hearing and advised me whenever - i t was necessary. F i n a l l y , I would l i k e to thank a l l my colleagues i n the parasitology laboratory f o r t h e i r support i n t h i s work. 1 I. INTRODUCTION The family Hemiuridae (Phylum Platyhelminthes: Class Trematoda) comprises digenetic trematodes occurring almost e n t i r e l y i n the g a s t r o - i n t e s t i n a l t r a c t of marine f i s h . A smaller number of these parasites i s found i n the buccal c a v i t y and stomach of amphibians, while fewer yet are located i n the lungs of c e r t a i n sea snakes. On the basis of t h e i r external morphology, the parasites i n the family may be grouped into99 subfamilies with t a i l s and l 6 without t a i l s , according to the c l a s s i f i c a t i o n of Yamaguti (1971). A l l but two of 3b genera of the t a i l e d hemiurids are found i n the stomach. In the t a i l e d f l u k e s , the body.is divided into an a n t e r i o r , broader soma and a p o s t e r i o r , somewhat narrower, t a i l - l i k e ecsoma which can be re t r a c t e d completely into the soma. The t a i l - l e s s hemiurids have no ecsoma. The soma and the ecsoma are both invested with an outer layer c a l l e d the tegument. While the soma tegument may be smooth, annulated, serrated or scaled, the ecsoma tegument i s smooth or occass i o n a l l y annulated. The soma accomodates the o r a l and v e n t r a l suckers and a l l of the reproductive organs (testes, vas deferens, pars p r o s t a t i c a , seminal v e s i c l e , sinus sac; ovary, oviduct, Mehlis' gland, v i t e l l a r i a , uterus). The digestive organs and the excretory 2 system begin, in,the soma but terminate i n the ecsoma. The ecsoma thus contains only the terminal portion of the digestive caeca;'' and the excretory bladder. In some species, the uterus may descend into the ecsoma. Although a few authors have speculated on the functions of the ecsoma, apparently basing t h e i r conclusions on observations of the hemiurids, the functions of f t he ecsoma s t i l l remain" unknown. The presence of the parasite i n the stomach offthe f i s h makes the ecsoma e s p e c i a l l y s i g n i f i c a n t . The present research was therefore undertaken to determine the nature and possible functionsoof f t he ecsoma. It was assumed that i n addition to the obvious differences between the soma and the ecsoma, h i s t o l o g i c a l sections and histochemical s t a i n i n g reactions would reveal further d i s s i m i l a r i t i e s between the two parts. In p a r t i c u l a r , the p o s s i b i l i t y that the tegument i n the soma and i n the ecsoma were d i f f e r e n t , and therefore functioned d i s t i n c t l y i n the ecsoma, was examined. The premise was the molecules and ions from the surrounding media passed across the tegument of the ecsoma, and thus the l a t t e r functioned i n the uptake of nut r i e n t s . T. The premise was based on the assumption that the tegument of the hemiurids was s i m i l a r to the tegument of most other digenetic trematodes investigated to date. Except f or one trematode specimen, a l l other species of the trematodes examined revealed that the tegument functioned i n the uptake and secretion of metabolites. The p h y s i o l o g i c a l l y active nature of the trematode tegument i s now c l e a r l y established''•(Smyth, 1°66; Erasmus, 1972). Evidence of i t s absorptive nature comes from electron microscope studies and from studies with l i g a t e d worms incubated i n nutrient media as well as from autoradiograph studies. Histochemical studies support the concept of the tegument as a metabolically active structure. In order to te s t the hypothesis that there was a s t r u c t u r a l and f u n c t i o n a l difference between the tegument of the soma and the ecsoma, and that amino acid could be transported across the ecsomal tegument, histochemical studies were followed by experiments i n which worms were incubated i n a medium containing radioactive g l y c i n e . 4 I I . REVIEW OF LITERATURE a) The Soma and the Ecsoma The soma and the ecsoma have heen c a l l e d d i f f e r e n t names by various authors. Looss (l n07) ( c i t e d by Dawes, 1947; Skrjabin, 1964), applied the term 'soma' to the anterior part and 'abdomen' to the post e r i o r region of the hemiurid. N i c o l l (1915) used the terms 'soma' and 'ecsoma' or 'appendix.' Dawes (1956) referred, to the two parts as 'soma' and 'ecsoma' while Skrjabin (1964) preferred using 'body proper' and 'tail-appendage.' Yamaguti (l97l) termed them• the 'body' and ' t a i l ' r e s p e c t i v e l y . In t h i s essay/ the terms 'soma' and' ecsoma* w i l l be used f or the anterior and the posterior regions r e s p e c t i v e l y . The ecsoma was considered to be the surviving member of the c e r c a r i a l t a i l by M o n t i c e l l i ( l 8 9 l ) , as c i t e d by Dawes (19-^7). According to Pratt (1898), M o n t i c e l l i ' s suggestions were based on the observations of Willemous-Suhm (1871). The l a t t e r author observed that immature Apoblema possessed an ecsoma when i t was a c e r c a r i a , and even when advanced beyond the c e r c a r i a l stage. The ecsoma as therefore the " s t r u c t u r a l counterpart of the cercarien t a i l " ( M o n t i c e l l i , 1891, c i t e d i n P r a t t , 1898). M o n t i c e l l i claimed that the two appendages were homologous. ; Both M o n t i c e l l i and Willemous-Suhm deduced that the ecsoma and the c e r c a r i a l t a i l were s i m i l a r ,.because the two functioned i n locomotion. According to M o n t i c e l l i (op. c i t . ) the worm l i v e d 5 a free-swimming existence i n copepod communities a f t e r emerging from the metacercarial stage, and used i t s ecsoma as a locomotor organ. Pratt (1898) described an immature hemiurid p a r a s i t e , Apoblema appendiculatum, i n great d e t a i l . The ecsoma was one-third the length of the soma. The tegument covering the soma and the ecsoma was 4 pm t h i c k . Stains revealed two layers i n the tegument. Lander (1904) could not d i s t i n g u i s h t h i s b i - l a y e r i n the tegument of Hemiurus crenatus i n which the tegument was 3.6 Lun ~$hi"ek. "The tegument of the soma i n the specimens described by Pratt (1898) and Lander (.1904) bore a grooved or 'ringed' appearance. This was i n contrast to the smooth tegument i n the ecsoma. Pratt (1898) describ/ed a columnar epithelium outside the tegument of the ecsoma i n h i s immature hemiurid specimen. This epithelium was shed i n the adult types. Pratt (1898) could not determine the arrangement of the muscle layers beneath the tegument. But he quoted Juel (1890) and said that an outer l o n g i t u d i n a l muscle layer was followed by an inner, c i r c u l a r l a y e r . This was the reverse of the arrangement of muscle layers i n the soma. Lander (1904) found the muscle arrangement i n the ecsoma of Hemiurus crenatus to be s i m i l a r to that described by J u e l (1890). Pratt (1898) recognized the s i m i l a r i t y between t h i s arrangement and that of the muscle layers i n the excretory v e s i c l e . He therefore considered the ecsoma to be a modified excretory v e s i c l e , and Lander (1904) accepted h i s views on the o r i g i n of the ecsoma. Both the above authors described parenchymal muscle f i b r e s i n t h e i r respective worms. In the ecsoma these muscle f i b r e s formed the powerful r e t r a c t o r muscles. Lander (l90'4) also described a layer of diagonal muscle f i b r e s i n the soma; these were absent i n the ecsoma. The parenchyma i n Apoblema appendiculatum consisted of 'highly vacuolated t i s s u e with n u c l e i lodged near the c e l l w a l l ' (Pratt, 1898). In Hemiurus crenatus the parenchyma was modified into a peripheral granular l a y e r , a v e s i c u l a r parenchyma between the various organs, and a c e l l u l a r sheath around the i n t e s t i n a l caeca i n the ecsoma (Lander, 190-U). Beside the parenchymal and muscle c e l l s , Pratt (1898) described submuscular c e l l s . These submuscular c e l l s were located beneath the c i r c u l a r and l o n g i t u d i n a l muscles in.the soma, but were absent i n the ecsoma. Three?: types of c e l l s were found by Lander (1904) i n the parenchyma. Type 1 c e l l s formed a syncytium i n the p e r i p h e r a l granular layer of the parenchyma. Nuclei were clustered at times and surrounded by a mass of protoplasm. Type 2 c e l l s wire l a r g e r , . pyriform or spindle-shaped, but were fewer than Type 1 c e l l s . Type 3 c e l l s were giant c e l l s and granular i n appearance. Type 2 and 3 c e l l s were both located i n the peripheral parenchyma. Pratt (1898) argues that the submuscular c e l l s secrete material.to the surface of the tegument to protect the worm against the enzymes from the host. Modern day electron microscope preparations 7 support Pratt's speculations (see Lumsden, 1975, for review). Pratt (1898) gives not. functions f o r the syncytium of c e l l s i n the perip h e r a l parenchyma. Lander (1904) d i d not think any of the c e l l types he described were glandular i n nature. The Type 1 and Type 2 c e l l s probably functioned i n forming myoblasts and muscular elements, and parenchyma " muscle f i b r e s r e s p e c t i v e l y . He suggested no function f o r the giant c e l l s . While Lander (1904) agreed with Pratt (1898) that the ecsoma might have arisen from an excretory v e s i c l e , he did not ascribe any function to the ecsoma. Pratt's views that the ecsoma arose as a part of the excretory system was shared by Looss (1907), according to Dawes (l9^7)rand Skrjabin (1964). But Looss (op. c i t . ) r e j e c t e d M o n t i c e l l i ' s claimsthat the ecsoma was the remnant of a c e r c a r i a l t a i l . Instead, Looss suggested from h i s observations that ( i ) the ./ tegument covering the soma was t h i c k e r than that covering the ecsoma and therefore was a protective material. The thickness of the tegument i n the soma d i d not permit the absorption of food or function i n r e s p i r a t i o n . Therefore ( i i ) the ecsoma, covered by a thinner • .•:,? tegument, performed absorptive and r e s p i r a t o r y functions. Because the ecsoma was capable of r e t r a c t i o n and p r o t r a c t i o n , i t withdrew into the soma during periods of a c i d i t y i n the stomach. Under more favourable conditions, the par a s i t e protracted i t s ecsoma and continued -the function of absorption and r e s p i r a t i o n . 8 b) C r i t i c i s m of the various theories M o n t i c e l l i ' s (1891) deduction that the ecsoma formed the surviving member of the c e r c a r i a l t a i l has been c r i t i c i z e d by Pratt (1898).--who contended that M o n t i c e l l i based h i s conclusions on the data published by Willemous-Suhm (1871), and not on the r e s u l t s of his own i n v e s t i g a t i o n . Pratt (1898) found the ecsoma i n the immature worm appeared as a sac or v e s i c l e within the pos t e r i o r end of the soma. He argued that the ecsoma d i d not appear 'as a prolongation of the soma whereas the c e r c a r i a l t a i l of digerietic worms did come out as an elongation of the c e r c a r i a l body. The ecsoma appeared, instead, to be protruded out through the terminal pore at the posterior end of the soma. Hence i t could not be likened to the c e r c a r i a l t a i l ( P r a t t , I 8 9 8 ) . M o n t i c e l l i ' s theory that the ecsoma i s a homologue of the c e r c a r i a l t a i l can now be countered with present-day data. The ecsoma could not have ar i s e n as an extension of the c e r c a r i a l t a i l because i n digenean trematodes the c e r c a r i a l t a i l i s shed a f t e r i t has served i t s function as a locomotory organ and the c e r c a r i a has found i t s next host (Erasmus, 1972) . Another argument against M o n t i c e l l i ' s explanation i s based on the biology of the hemiurid c e r c a r i a . The l a t t e r i s a cystophorous type. The c e r c a r i a l body proper i n t h i s type ^ i s attached to a greatly modified c e r c a r i a l t a i l . The t a i l i s a v e s i c u l a r chamber (also c a l l e d a t a i l - v e s i c l e by Rothschild, 1 9 3 8 ) , and to t h i s are attached two to four appendages c a l l e d the d e l i v e r y tube, ribbon 9 of S i n i t s i n , "Sultan's" plume of S i n i t s i n , and Phrygian cap (handle). P r i o r to becoming a metacerearial cyst, which i s the next stage of the development, the c e r c a r i a l body proper withdraws into the chamber, loses i t s attachment to the v e s i c u l a r chamber ( K r u l l , 1935; Rothschild, 1938; Hussey, 1941) , and escapes through the d e l i v e r y tube. The ecsoma i s a sac, appearing within^the hind part of the soma, and i s not protruded out of the soma when the c e r c a r i a leaves i t s intermediate host (Pratt, I898). Lebour (1923) found that the "abdomen" (ecsoma) i n immature Hemiurus communis was invaginated into the soma at the p o s t e r i o r end. These worms, located within a copepod, escaped out of t h e i r host with the posterior end ,pf the soma f i r s t , and with the ecsoma s t i l l i n the invaginated state. Thus, Pratt's (1898) observations are confirmed by Lebour (1923). M o n t i c e l l i ' s argument that the ecsoma was used as a locomotory organ to a t t r a c t a host could not be v e r i f i e d by Pratt (1898). The l a t t e r author observed free-swimming worms but found no locomotory behaviour j i n the ecsoma. Neither did Lebour (1935) f i n d that immature Hemiurus communis showed extended ecsomas a f t e r these had escaped from copepod hosts. I t i s therefore l i k e l y that the ecsoma i s not a locomotory organ. Pratt's (1898) inferences are more consonant with the embryo-l o g i c a l development of the f l u k e , and h i s suggestion that the ecsoma i s a part of the excretory v e s i c l e might be supported by some present-day data on the embryological development of .'.the-excretory "' system. 10 Hussey (1941, 1943) and Kuntz (1950, 1951, 1952) examined the development of the excretory system i n d i f f e r e n t types of cercariae, including cystophorous types. Their investigations showed that the excretory bladder i s formed i n the body proper of the c e r c a r i a , and when a c e r c a r i a l t a i l i s developed, the bladder descended into the t a i l . La Rue (1957) divided excretory bladders into ( l ) non-e p i t h e l i a l types and (2) e p i t h e l i a l types. In n o n - e p i t h e l i a l types a retrogression of the excretory v e s i c l e occurs from the c e r c a r i a l t a i l into the body proper. Eventually, the c e r c a r i a l t a i l i s l o s t , arid an excretory pore appears where the t a i l was attached to the c e r c a r i a l body. In the e p i t h e l i a l type of excretory bladder, meso-dermal t i s s u e occurs between the primary excretory ducts i n the hind portion of the body proper. The mesodermal tissues "mold" into the excretory ducts and form the excretory bladder (La Rue, 1957). In the superfamilies Hemiuroidea,dn Plagiorchiodea the c e r c a r i a l ' t a i l i s formed by a "molding" of the post e r i o r part of the body, and the excretory bladder from the body proper i s extended as an excretory duct i n t o the c e r c a r i a l t a i l ( F i g . l ) . In other superfamilies (Plagiorchiodea and Allocreadidoidea), the hind part of the body proper i s not "molded" to form the t a i l , and+-ther€for.erino--portion of the excretory bladder i s moved into the t a i l (La Rue, 1957). The cystophorous type of c e r c a r i a of the hemiurid parasites possesses the e p i t h e l i a l type of excretory bladder (La Rue, 1957). As mentioned e a r l i e r , the body of the cystophorous c e r c a r i a detaches i t s e l f from the t a i l when i t draws into the v e s i c u l a r chamber. 11 Hence;, there i s no further connection with the t a i l . From*.the foregoing discussion i t can be-seen that the soma has no connection with the c e r c a r i a l t a i l , and hence M o n t i c e l l i 1 s (1891) conclusions cannot be true. The discussion also shows that the excretory bladder, formed by t i s s u e of mesodermal o r i g i n , l i e s within the hind part of the c e r c a r i a l body proper. Pratt (1898) and Lebour (1923) both observed that the ecsoma i n t h e i r young appendiculate worms l a y within the hind part of the soma. It may therefore be speculated that following the formation of the e p i t h e l i a l type of excretory bladder, the ecsoma arose as an outgrowth of tissues of mesodermal o r i g i n which also contributed to the development of the excretory bladder. Hence i t i s more l i k e l y that Pratt's (1898) explanation f o r the o r i g i n of the ecsoma i s true. Pratt (1898) also pointed out the s i m i l a r i t y that exists between the arrangement of the muscle layers i n the ecsomao and excretory bladder. This furnishes further evidence that the ecsoma and the excretory bladder have a common o r i g i n . While the ecsoma may have originated as a part of the excretory system, whether or not i t ; functions as an excretory organ 1 i s open to question. The ecsoma possesses well-developed excretory ducts and a bladder which leads to the outside by an excretory pore. I t could be possible f o r the ecsoma to excrete a d d i t i o n a l l y through the tegument; however, there i s no evidence i n the l i t e r a t u r e that teguments carry out t h i s function. 12 The p o s s i b i l i t y that the ecsoma functions i n r e s p i r a t i o n according to Looss (1907) may be true, but there i s no evidence for t h i s i n the l i t e r a t u r e . Because trematodes lack an organized r e s p i r a t o r y system, i t i s possible that r e s p i r a t i o n occurs on the surface of the tegument (Erasmus, 1972), and only the presence of mitochondria i n the tegument suggests t h i s . Since no r e s p i r a t o r y enzymes have been detected on the tegument, i t cannot be s a i d with c e r t a i n t y that the tegument functions i n r e s p i r a t i o n . It i s more l i k e l y that since parenchyma i n other worms is.' known to be storage spaces f or nutrients (von Brand, 1973), the ecsoma, with i t s large parenchymal space, could function i n the storage of carbohydrates :and proteins. Although these nutrients could be taken throueht.the mouth and passed into the ecsoma through i,ne moutn the caeca, i t i s possible f o r the nutrients to be absorbed through the tegument of the ecsoma and stored i n the parenchyma. The function of absorption could be c a r r i e d through the tegument because the tegument i s now known to be a p h y s i o l o g i c a l l y active s y n c y t i a l layer (Lumsden, 1975). c) The Tegument The external covering of the parasite i s the tegument. It l i e s above the c i r c u l a r muscle layer that i s o r d i n a r i l y seen i n cross-sections of platyhelminth t i s s u e s . P r i o r to electron microscope studies, the tegument was c a l l e d t h e ' c u t i c l e , ' and was considered a n o n - l i v i n g secretion of the subeutlicular c e l l s beneath the muscle 13 l a y e r s . E lectron micrographs reveal that the tegument i s a l i v i n g , and- therefore, dynamic^, cytoplasmic extension of the c e l l s beneath the muscle l a y e r s . It i s an e p i t h e l i a l syncytium ('Lumsden, 1975), bounded on theoutermost surface by a plasma-membrane. Several reviews have been published on^the tegument of trematodes. Lee (1966, 1972) and Lumsden (1975) c i t e u l t r a s t r u c t u r a l and biochemical studies on the tegument. The reviews of Mettrick and Podesta (1974), Pappas and Read (1975),- and the Symposium on B i o l o g i c a l Surfaces (Tr. Am. Micr. S o c , 1975, 94:449-55*0 point to the importance of tegumental surface membranes i n b i o l o g i c a l transport mechanisms. Senft (1959), Senft et_ al.(1961) published the f i r s t electron microscope study of the tegument, using Schistosoma mansoni as the experimental material. Threadgold (1963a, b) gave a more d e t a i l e d d e s c r i p t i o n of the tegument i n F a s c i o l a hepatica.,- (Fig. 2). Studies on F_. hepatica (Bjorkman and T h o r s e l l , 1964a), Haematoloechus  medioplexus (Burton, 1964), Gorgoderina sp. (Burton, 1966; B i l s and Martin, 1966), Acanthoparyphium spinulosum (Erasmus and Ohman, 1965), Cyathocotyle bushiensis, Apatemon g r a c i l i s minor and Diplostomum  phoxini (Erasmus 1967, 1969) a, b, c, 1970), Posthodiplostomum  minimum (j3ogitsh and Aldridge, 1967), and Schistosoma mansoni (Morris and Threadgold, 1968) show that the basic features of the tegument of these trematodes are s i m i l a r to those of F a s c i o l a hepatica. 14 The thickness of the tegument varies from 2 to 6 /um i n A. spinulosum to 15 ,um i n hepatica. Depending upon the parasite being studied, the topography of the tegument may show crypts, v a l l e y s , v e s i c l e s , m i c r o v i l l i and lamelliform p r o j e c t i o n s , thus increasing the surface area and the absorptive capacity. Mitochondria, endoplasmic reticulum, Golgi apparatus, small v e s i c l e , ovoid vacuoles and spines are found i n the tegument. But v a r i a t i o n s occur between d i f f e r e n t species of p a r a s i t e s . In C. -bushiensis, Erasmus (1967) found secretory bodies i n the tegument which are v i s i b l e i n few other p a r a s i t e s . The tegument contains a small amount of glycogen, some npn-glycogen polysaccharides and sometimes l i p i d s (Lee, 1966; Erasmus, 1972). The major component appears to be a c i d mucopolysaccha-rides and a Periodic Acid S c h i f f (PAS) p o s i t i v e , diastase r e s i s t a n t substance (Berthier, 1954; Monne, 1959; L a i and Srivastava, i960; Bjorkman et a l . , 1963; Erasmus and Ohman, 1963; Rothman and Elder, 1970; Stein and Lumsden, 1973). Several workers have shown acid and a l k a l i n e phosphatase a c t i v i t y i n trematode teguments (Dusanic, 1959; Lewert and'Dusanic, 196l; Robinson, 196l; Nimmo-Smith and Standen, 1963; Halton, 1967; Bogitsh, 1966b; Rothman, 1968; Erasmus, 1972). Some fu n c t i o n a l considerations have been proposed f o r the acid mucopolysaccharides and the enzymes. Monne (1959) suggested that the a c i d mucopolysaccharide acts as a s h i e l d against the host's h i s t o l y t i c enzymes. Rothman and Elder (1970) opined that 15 trematodes adsorb digestive enzymes while Crompton (1973) suggested these may r e s i s t the host's enzymes rather than i n h i b i t them. Histochemical stains have been used to demonstrate acid and al k a l i n e phosphatases, and to l i n k the presence of these enzymes i n the tegument to .an "extracorporeal digestive function" (Lumsden, 1975). That i s , these and other secretions l y s e the host material around the p a r a s i t e , thus allowing the worm to ingest material through the tegument. Although phosphatases function i n other, a c t i v i t i e s , a c i d phosphatases are b a s i c a l l y r e l a t e d to the process of digestion ( P i t t , 1975). Food may be taken i n by pinocytosis and digested by the acid phosphatase contained i n lysosomes, or microsomes. A l t e r n a t e l y , lysosomes may secrete acid.phosphatase and , ;digest food by exocytosis (Smith and Farquhar, 1966). According to Threadgold (1968), phosphatases probably dephosphorylate phosphory-'. t l a t e d compounds that cross plasma membranes. The lamellae on the teguments, and the p h y s i o l o g i c a l l y active nature of these combined together, i n d i c a t e an absorptive function for the tegument. The evidence for t h i s i s borne out by a number of studies with l i g a t e d parasites incubated i n rad i o - a c t i v e material. Mansour (1959) l i g a t e d F a s c i o l a hepatica at the o r a l sucker and showed that the worm incorporated glucose through the tegument. Ligated hepatica also absorbed cycloleucine (l-amino cyclopentane-l-carboxylic a c i d ) , arginine, p r o l i n e and methionine ( i s s e r o f f and Read, 1969), alanine and glutamic acid (Kufcelec f_ ". 16 and Ehrlic,.1963) v i a the tegument. Fascioloides absorbed c y c l o -leucine, arginine, p r o l i n e and methionine s i m i l a r l y ( I s s e r o f f and Read, 1969). Other uptake studies were reported by Nollen (1968 a, b) for' Fhilopthalmus megalurus and Gorgoderina attenuata; I s s e r o f f et a l . (1972) for S. mansoni; Fripp (1967) for S. hematobium; Bhatti and Johnson (l97l) for A l a r i a marcianae; inrt Parkening and Johnson (1969), Pappas (1971) and McCracken (1972) for Haematoloechus medioplexus; and Asch and Read (1975) f o r S. mansoni. However, Shannon and Bogitsh (1971) and Bogitsh and Krupa (l97l) concluded that the tegument of Megalodiscus temperatus was impermeable to sugars and amino acids. IT I I I . MATERIALS AND METHODS a) Observations i n the f i e l d The hemiurid trematode, Tubulovesicula l i n d b e r g i (Layman, 1930) Yamaguti, 1934 was chosen for the study. The worms (Fig.*3) used i n t h i s research measured 2.31-4.38 x 0.627-1.32 mm. i n the soma region and 0.957-2.3^ x 0.36-0.72 mm. i n the ecsoma region. The parasite i s e a s i l y recognized by a parss p r o s t a t i c a which i s about 1.0 mm. long and by i t s long, sinuous, tubular seminal v e s i c l e . I t occurs i n the stomach of a number of d i f f e r e n t f i s h , e s p e c i a l l y i n the pleuronectids d i s t r i b u t e d around the rim of the P a c i f i c Ocean. Arai (1969) reported i t i n nine species of f i s h i n Burke Channel, B r i t i s h Columbia, none of which were pleuronectids. Although A r a i (1969) found another t a i l e d hemiurid species i n pleuronectids, I was able to f i n d only . .: T. l i n d b e r g i i n the f l a t f i s h i n the Georgia S t r a i t s , B r i t i s h Columbia, a f t e r examination of a large number of host specimens (Table Ia, l b ) . F i s h were caught by otter trawl and when possible brought a l i v e to the laboratory. They were kept i n holding tanks i n sea water. Fis h were removed p e r i o d i c a l l y as needed, and k i l l e d by a sharp blow on the head. Because many of the f i s h , e s p e c i a l l y Parophrys vetulus, died during transportation from the catch area to the laboratory, many f i s h were dissected aboard the trawling v e s s e l . The stomach was removed • a 18 removed and placed i n marine t e l e o s t s a l i n e (Hoar and Hickman, 1975) and brought to the laboratory. Parasites were removed thereafter and r e f r i g e r a t e d at 15 °C i n p e t r i dishes. The saline s o l u t i o n was replaced by fresh' s a l i n e every two days. Under such treatment, parasites l i v e d f o r as long as ten days. b) Location and 'posture of flukes i n f i s h hosts In f i s h which died before reaching the laboratory, parasites were found on the body surface of the f i s h . When the stomachs and int e s t i n e s of these f i s h were examined, no parasites were found i n them. , During one f i s h i n g t r i p , on-the-spot observations were made on the parasites within the stomach of the f i s h to determine whether (i) the ecsoma was protracted or re t r a c t e d ; ( i i ) thet,protraetion or r e t r a c t i o n , i f any, was dependent upon an empty stomach, or :,upon a f i l l e d stomach; ( i i i ) prcptraction or r e t r a c t i o n was dependent ,upon the pH. The observations were further intended to reveal the (i ) p o s i t i o n occupied by the parasite i n the stomach, and ( i i ) the or i e n t a t i o n of the mouth and ecsoma with respect to the oesophageal opening of the f i s h host. The stomach of several species of f i s h was s l i t open lengthwise sho r t l y a f t e r the f i s h were removed from the net. The pH was determined by applying pH i n d i c a t o r s t r i p s (PAN PEHA, VWR S c i e n t i f i c , San Francisco, C a l i f o r n i a ) to the eavityh of the stomach immediately a f t e r i t was opened. The parasites and the stomach contents were observed using a hand lens. 19 c) Observations in.the laboratory Several observations were made on the parasite and i t s p o s i t i o n , and that of the ecsoma i n the stomach of f i s h that were s a c r i f i c e d i n the laboratory. Stomachs were s l i t open l o n g i t u d i n a l l y and the parasites were observed under an Olympus d i s s e c t i n g microscope. The extension and pBOtraction of the ecsoma was noted. Parasites were removed from the stomach and observed for long periods i n marine t e l e o s t saline to determine i f there was a regular pattern of movement of the ecsoma. d) E f f e c t of pH on the ecsoma Mackenzie and Gibson (1970) found that the pH i n the stomach was a c i d i c when Platichthys flesus was fed and neutral when i t was empty. To see whether such pH changes also a f f e c t e d the parasite and i t s ecsoma, the following experiment was c a r r i e d out. Worms were removed from t h e i r p e t r i dishes and washed i n fresh marine t e l e o s t s a l i n e . At the beginning of the experiment only those worms were chosen which had t h e i r ecsomas i n the ret r a c t e d condition. Worms were then put i n phosphate buffered s a l i n e solutions which were adjusted for the pH values by using IN HCl or IN NaOH. Eleven p e t r i dishes, with solutions ranging from pH 1 "to pH 11 were set up. Three parasites were put into each dish. The parasites were observed over a period of time and frequency/with which they protracted t h e i r ecsomas over t h i s period of time was :i noted. The experiment was conducted at 15 °C. 20 e) E f f e c t of s a l i n i t y on the ecsoma Data from Mackenzie and Gibson (1970) showed that the osmotic pressure i n the stomach of Platichthys flesus v a r i e d . It was therefore preBumed that the stomach f l u i d i n the present f i s h ,,hosts might be subjected to osmotic pressure changes, and t h i s might a f f e c t the p r o t r a c t i o n ( and. r e t r a c t i o n of the ecsoma. The s a l i n i t y of marine t e l e o s t s a l i n e (0.78$) was used as a base fig u r e and was considered to be a 100$ s o l u t i o n . By adding so<k«.-m. • , ^ i nn c h l o r i d e , the s a l i n i t y was r a i s e d . D i l u t i n g the solution decreased the s a l i n i t y . Solutions were prepared to 2Xvel.8X, 1.6X, 1.4X, 1.2X, 0.8X, 0.6X, 0.4X, and 0.2X the concentration of the marine t e l e o s t s a l i n e . D i s t i l l e d water was used to represent no s a l i n i t y . The 0.78$ marine t e l e o s t s a l i n e , which was considered to be a 100$ s o l u t i o n , was used as a control s o l u t i o n . Worms, three i n number, were placed i n the s o l u t i o n s i i n stender dishes, with t h e i r ecsomas re t r a c t e d . They were observed every 15 minutes over a period of time and the p r o t r a c t i o n of the ecsoma was noted at 15 °C. f) P r o t r a c t i o n of the ecsoma F i e l d observations showed that the ecsoma was retracted most of the time (Table I I ) . The ecsoma was also found r e t r a c t e d i n specimens that were removed from the stomach and l e f t i n marine t e l e o s t s a l i n e . Several solutions were therefore t r i e d to induce a chemical p r o t r a c t i o n of the ecsoma. 21 Worms, with t h e i r ecsoma r e t r a c t e d , were l e f t i n three solutions: (i ) 0.75 s o l u t i o n of t r i c a i n e methanesulfonate; ( i i ) saturated magnesium chloride d i l u t e d to 50% by adding marine.teleost s o l u t i o n , and i n ( i i i ) marine t e l e o s t s a l i n e to which a few drops of menthol were added. Worms were also observed i n d i s t i l l e d water, i n dechlorinated tap water cooled to near-freezing temperature, and i n saline solutions cooled to below 10 °C. A l l these procedures gave negative r e s u l t s . F i n a l l y , i n the method adopted, the parasite was put on a glass s l i d e and gently pressured under a cover s l i p with the index fingers and thumbs from two sides. This caused a pr o t r a c t i o n of the ecsoma to a small degree. Further extension was achieved by holding the end of the ecsoma with a watch repa i r e r ' s forceps as near to the excretory pore as possible and p u l l i n g i t out gently. g) Morphological, h i s t o l o g i c a l studies To study theggeneral morphology, the parasites were f i x e d i n warm alcohol-formalin-acetic a c i d s o l u t i o n (Meyer and Penner, 1962), or i n 10% neutral formalin (Pearse, 1968) under cover s l i p pressure. Sections for his t o l o g y were obtained by dehydration and embedding fi x e d parasites i n p a r a f f i n wax and cuttin g them at 6 urn on a AO Spencer Microtome. Sections, 6 yum t h i c k , were also obtained by cu t t i n g unfixed worms on a IEC-CTF Microtome Cryostat (International Equipment Company). 22 In view of the d e l i c a t e nature of the worm, cryostat sections were prepared the following way: a small amount of cryogel was placed on the chuck and l e f t i n the cryostat cabinet to freeze from the periphery towards the centre. Before the cryogel i n the centre could freeze, the chuck was removed to room temperature. The p a r a s i t e , with s l i g h t l y extended ecsoma, was l a i d with the ecsoma f i r s t on the cryogel that was frozen and gently p u l l e d across the unfrozen c e n t r a l surface. I f placed intthe cabinet o f t the cryostat, the worm became embedded i n the cryogel. The frozen cryogel was sectioned along with the worm and picked up on cover s l i p s . These sections were air. d r i e d b r i e f l y , f i x e d and stained. Whole specimens were stained i n Semichon's carmine or i n Har r i s ' haematoxylin (Meyer and Penner, 1962). Microtome and cryostat sections were r o u t i n e l y stained i n :Harris' haematoxylin and i n 1% aqueous eosin. Whole mounts and sections were dehydrated i n alcohols and cleared either,-ri-rv graded series of alcohol-xylene mixtures or i n cedarwood o i l . Clove o i l was also used to clear some specimens. Cleared specimen were mounted i n Permount (Fisher S c i e n t i f i c Company). The following histochemical s t a i n i n g reactions were c a r r i e d out: l ) Carbohydrates: A l c i a n blue, Periodic Acid S c h i f f (PAS), PAS with diastase, toluidene blue ( C u l l i n g , 1963; McManus and Mowry, 1965; Pearse, 1968). 23 2) Proteins: Dinitro'fluorobenzene (DNFB), Chevremont-Frederick method for -SH groups (Chayen, Bitensky and Butcher, 1 9 7 3 ) . 3 ) L i p i d s : Sudan black ( C u l l i n g , 1 9 6 3 ; McManus and Mowry, 1 9 6 5 ; Pearse, 1 9 6 8 ) . h) Enzyme histochemistry Histochemical t e s t s were used to determine the presence of two enzymes, a l k a l i n e and acid phosphatase, which aree concerned with digestive processes (Lumsden, 1 9 7 5 ) . Gomori's cobalt sulphide method for a l k a l i n e phosphatase and Gomori's lead sulphide method for acid phosphatase ( C u l l i n g , 1 9 6 3 ) were employed. P a r a f f i n embedded and cryostat sections were incubated i n the Gomori medium and r e s u l t s , i n the case of acid phosphatase, were v i s i b l e i n 1 5 minutes a f t e r incubation. In the controls used, ( i ) sections were incubated i n Gomori's medium minus the sodium glycerophosphate; ( i i ) sections were b o i l e d i n water and incubated i n Gomori's so l u t i o n ; . ( i i i ) sections were treated in. 5$ t r i c h l o r o a c e t i c a c i d and then incubated i n Gomori's s o l u t i o n . In anotherttest, a c i d phosphatase was determined on l i v e T. l i n d b e r g i . The worms were pressed under a cover s l i p to extend - the ecsoma. In the extended p o s i t i o n the ecsoma was not allowed to r e t r a c t by using a s l i d e as added weight on top of the cover s l i p . The worms were then incubated In a Gomori s o l u t i o n f o r 1 5 minutes. Controls were used by omitting the glycerophosphate from the incubating medium and incubating the worm for 1 5 minutes. 24 i ) Uptake of G 1 4 glyc me To t e s t the hypothesis that the tegument of the ecsoma was capable of absorbing amino acids from the surrounding medium, whole worms i n which the ecsomas were l i g a t e d were incubated i n 14 marine t e l e o s t saline containing C g l y c i n e . The ecsoma was protracted using c o v e r - s l i p pressure. Further extension of the ecsoma was achieved by gently p u l l i n g at the i ecsoma with a p a i r of watch repa i r e r ' s forceps. The procedure e n t a i l e d some i n j u r y to the ecsoma. Consequently, the t i p s of the forceps were applied as close to the excretory pore as poss i b l e . Using a strand of nylon sthread (Beldings 100% #50), the ecsoma was quickly l i g a t e d at a point nearest to the ecsoma-soma junction. Human h a i r was also used as a l i g a t u r e , and when i t was found that there was no differ e n c e i n the s c i n t i l l a t o r countings whether an ecsoma was l i g a t e d with a strand of thread or h a i r , h a i r was used by choice. A l i g a t u r e was t i e d at the pos t e r i o r end of the ecsoma, close to the excretory pore and above the region where the ecsoma was handled with the forceps. This prevented the p o s s i b i l i t y of glycine being taken into, the ecsoma through the excretory pore, as well as being taken through the area where the forceps might have damaged the tegument. Ligated worms were l e f t i n p e t r i dishes that were placed on top of i c e f o r 15 minutes ;.before incubation was c a r r i e d out i n the glycine-containing medium. 25 Worms were also l i g a t e d below the o r a l sucker region and at the junction of the soma-ecsoma, as well as ;' anterior to the excretory pore. The worms were incubated i n solu t i o n containing glycine i n order to determine whether amino acids could be transported through the soma and the ecsoma tegument. Such worms died within 30 minutes of l i g a t i o n , and hence t h i s experiment was abandoned. D i l u t i o n of C"*"^  g l y c i n e : Uniformly l a b e l l e d C"^ gl y c i n e , s p e c i f i c a c t i v i t y 102 mCi/mmole (New England Nuclear, Boston, Mass.) was used. Twenty m i c r o l i t r e s of the glycine ^solution was' d i l u t e d i n 2.0 ml of marine t e l e o s t s a l i n e and s t e r i l i z e d by adding 0.03 mg/ml of p e n i c i l l i n and 0.01 mg/ml streptomycin. The t o t a l concentration of glycine was thus 0.07 x 10 ^ mg/ml. Groups of f i v e worms were incubated i n 200 i l l of t h i s s o l u t i o n for periods of 1, 7, 15, 30 and 60 minutes..AAfterr-'incubation, worms werevrwashed i n a large volume of flowing marine t e l e o s t s a l i n e for one minute to remove any rad i o a c t i v e glycine adsorbed to the external surface. Worms were then put on a glass s l i d e and the l i g a t e d section of the ecsoma was excised with a s c a l p e l . The excised sections were mashed and broken up .with the blunt end of a glass rod i n 3.0 ml 95% ethanol, and the glycine was extracted for 2k hours. Aliquots of 1.0 ml of the 95% extract were put into 10.0 ml of f l u o r Formula 950 A (New England Nuclear, Boston, Mass.). The r a d i o a c t i v i t y was determined with an Isocap II S c i n t i l l a t i o n Counter. Internal standards were used to correct f o r quenching. 26 j ) Tests f o r p r o t e o l y t i c enzymes It was speculated that the ecsoma might also secrete p r o t e o l y t i c enzymes to break down proteins appearing i n the surrounding medium. Using ecsoma extracts prepared by homogenizing f i v e excised ecsomas i n marine-teleost s a l i n e , three methods were used to'detect p r o t e o l y t i c a c i t i v i t y . In the f i r s t method, 5 ]pl of the extract was placed on the emulsion surface of an exposed photographic f i l m , and allowed to incubated at 10 °C for 12 hours. The f i l m was washed i n tap water and observed f o r white, lysed areas. In the second procedure, egg-white was drawn up i n a 75 mm Yankee micro-hematocrit tube (Clay-Adams, Inc., N.Y.) and b o i l e d . This caused the egg-white to coagulate. One centimeter pieces of the tube were cut h and l e f t i n the ecsoma extract to incubate f o r 24 hours at 10 °C. P r o t e o l y t i c a c t i v i t y was indicated i n sections where the egg-white had disappeared. In the t h i r d technique, the.method of Jarumilinata and Maegraith (l n 6 l ) and Weal (l n 6 l ) were employed. A t h i n f i l m of 6% g e l a t i n was spread on a microscope s l i d e and allowed to dry. Ecsoma extract (5 ;ul) was placed on the g e l a t i n and incubated at 10°C for 24 hours. Slides were then f i x e d i n k% formalin f o r 5 minutes, • washed i n running water to remove the formalin, and stained i n Har r i s ' haematoxylin for 30 minutes. The s l i d e was blued i n tap water. Areas which had lysed d i d not s t a i n with haematoxylin while other areas d i d . 27 Of the three methods t r i e d above, the 6% g e l a t i n f i l m method produced the most e a s i l y detectable lysed areas, and was therefore adopted f or subsequent experiments. Two experiments were designed to see whether p r o t e o l y t i c enzymes were secreted through the tegument. F i r s t , an excised ecsoma was l i g a t e d a n t e r i o r l y and p o s t e r i o r l y with human h a i r and allowed to incubate i n 2.:0.j.ml of marine t e l e o s t s a l i n e at 10 °C for 24 hours.. Following incubation, the ecsoma was removed and 5 u l of tlie". extract placed on a 6% g e l a t i n layer as described above for 24 hours at 10 °C. Five m i c r o l i t r e s of marine t e l e o s t s a l i n e and 5 ^ 1 of 1% t r y p s i n were incubated s i m i l a r l y on two separate g e l a t i n layered s l i d e s . The s l i d e s were f i x e d , stained, washed and observed for l y s i s . Secondly, 6% s g e l a t i n s o l u t i o n was placed i n well s l i d e s and allowed to g e l . The l i g a t e d parasites were placed on+the gel surface and allowed to incubate at 10 °C for 24 hours. Well s l i d e s were f i x e d and stained as above-?. IV. RESULTS a) Observations i n the f i e l d The parasites that appeared outside the body of the dead f i s h were found on the white, nether surface of the f l a t f i s h and had crawled out the mouth of the hosts. The ecsoma was re t r a c t e d i n most cases. The r e t r a c t i o n was not however complete, and approximately one-tenth of the ecsoma was protruded outside of the soma i n the specimens that were observed. Table I l a indicates the pH values encountered i n the stomach range from 3 to 6. Most of the f i s h examined were Reinhardtius  hippoglossoides i n the Stewart Channel area. Of the 21 T_^  l i n d b e r g i observed, 18 were found at pH 6, and the re s t at pH k :and 5. Seventeen parasites were found i n a stomach that contained food while the remaining four occurred i n empty stomachs. The sizes of a l l these parasites were small i n comparison to the specimens examined from other species of f l a t f i s h i n other areas of the Georgia S t r a i t . .. The ecsoma was retracted completely i n IT specimens (see Table I l a ) . Thirteen of these were i n fed and four i n un-fed f i s h . The four specimens with protracted ecsomas were found i n f i l l e d stomachs none were i n the empty stomachs. The degree of 1 p r o t r a c t i o n was approximately h a l f of the length of the soma. Further observations showed that 15 of 21 T_j_ l i n d b e r g i occupied the cardiac section; 6 were i n the mid-section, and none was located i n the p y l o r i c region of the stomach (Table l i b ) . 29 Fourteen of the 21 parasites were oriented with t h e i r o r a l end towards the mouth opening of the f i s h , while four and three were dire c t e d l a t e r a l l y and p o s t e r i o r l y . The r e s u l t s are summarized i n i n Table l i d . Of the specimens with protracted ecsomas, two occupied the mid-section i n the stomach, and both were directed with t h e i r oral"iopening toward the p y l o r i c end. Two others occupied the anterior region of the stomach, with oralJopenings towards the oesophagus. b) Observations i n the laboratory In f i s h that were held i n c a p t i v i t y and opened i n l t h e laboratory, most of the parasites were oriented i n a l o n g i t u d i n a l axis i n the stomach. The o r a l opening was ^directed towardssthe oesophagus of the host i n 21 of 2k instances. Two were directed l a t e r a l l y and one p o s t e r i o r l y . Seventeen of these specimens occupied the cardiac section, while s i x were i n the mid-section, and one was i n the p y l o r i c section of the stomach ( T a b l e l l l e ) . Because f l a t f i s h d i d not feed i n c a p t i v i t y , the e f f e c t of food on the parasites i n the stomach could not be assessediihtt'h'eii.abor,atory. 95% of the T. l i n d b e r g i were observed with the ecsoma completely r e t r a c t e d into the soma. The ecsoma was drawn into the soma. The withdrawl l e f t a minute aperture at the posterior part of the soma. In protracted specimens., the ecsoma was never f u l l y extended. The ecsoma was protracted about one-tenth the length of the soma, 30 and remained for long periods i n t h i s p o s i t i o n . In a f.ew instances, the protracted ecsoma would he extended for 10-15 seconds to about h a l f the length of the soma, and then r e t r a c t e d again. Specimens l e f t i n marine t e l e o s t s a l i n e i n a p e t r i d i s h remained with t h e i r ecsoma retracted. It was observed .on various occasions that the ecsoma was extended f o r b r i e f periods, and retracted again. There was no p e r i o d i c i t y i n . t h i s movement. c) E f f e c t of pH on the ecsoma It i s evident from Table I I I that the extension of the ecsoma was not influenced by hydrion concentration. The ecsoma remained r e t r a c t e d i n acid and a l k a l i n e solutions of sea water made up i n the laboratory. The r e t r a c t i o n was complete and the ecsoma was f u l l y withdrawn into the soma. Where p r o t r a c t i o n was evident, i t was a f r a c t i o n of the t o t a l soma length. The a b i l i t y of the parasites to survive i n solutions of varying pH was demonstrated i n the same experiment. It i s clear that T. l i n d b e r g i cannot survive long at pH 3 and lower. In specimens that died at these pH values, th'e': soma tegument .was s e e T f l i . f i r s t to turn white and then granular. The granular covering was a t i times observed to be covered by an i r i d e s c e n t sheen. When specimens with extended ecsoma were-left i n solutions at pH 2, the ecsoma tegument turned granular before the soma tegument. Parasites were better able to survive for longer periods i n a l k a l i n e media. A f t e r 24 hours no ph y s i c a l e f f e c t s of a l k a l i n i t y 31 showed on the tegument. Control specimens l e f t i n neutral 0.1'}% s a l i n e survived for 24 hours without any apparent damage or r e t r a c t i o n of the ecsoma. d) E f f e c t of s a l i n i t y on the ecsoma.. . Parasites did not respond to changes i n s a l i n i t y and the ecsoma remained i n the r e t r a c t e d p o s i t i o n throughout the experiment (Table IV). However, the ecsoma was protracted within three hours i n worms which had died add which were l e f t i n d i s t i l l e d water. The appearance of the ecsoma i n these specimen was smooth and glassy white. Unlike specimens i n acid s o l u t i o n , they did not turn granular. Figure 4 shows that the tolerance of T^ _ l i n d b e r g i to s a l i n i t y changes i s high. Parasites died i n marine t e l e o s t .saline which had been concentrated to twice i t s normal salinity-,v»but+survived s a l i n i t i e s as low as 0.2X the concentration. e) Morphological and h i s t o l o g i c a l studies: - m e ( i ) The tegument: The whole mounts prepared by revealed.that the soma and ecsoma are covered by a tegument. The tegument i s thicker i n the ecsoma than i n the soma region and measurements for these are given i n the. sect'oion below. The soma tegument i s smooth whereas the ecsoma tegument i s annulated. The tegumsnt does not s t a i n with Semichon's carmine and only s l i g h t l y with H a r r i s 1 haematoxylin. B a l l o o n - l i k e and bubble-like i n f l a t i o n s (Fig) 5) were observed on the surface of the ecsoma tegument. These were 32 observed not to s t a i n with carmine or haematoxylin. The i n f l a t i o n s d i d not occupy the whole length of the ecsoma and were seen/in some parts of the ecsoma only. E s p e c i a l l y at the posterior end, there appeared an extra layer of ecsoma tegument (Fig. 6). This was found i n only seven worms examined from d i f f e r e n t host species. Whole mounts also revealed that the parenchymal c e l l s i n the ecsoma were more numerduss-.than i n the soma. No actual counts were made. The n u c l e i of these c e l l s stained blue with haematoxylin. The observations on the whole mount were confirmed and r e i n f o r c e d i n h i s t o l o g i c a l sections. In a d d i t i o n , more differences become apparent between the tegument i n the soma and the ecsoma. The tegument of the soma runs down the length of the soma and tucks inwardly to meet the tegument of theddescending portion of the ecsoma (Fig. 7)- I t thickens to some extent before thinning out to merge with the tegument of the ecsoma. A c l e f t appears i n the tegument where the soma and the ecsoma meet. The tegument of the soma (Fig. 8) i s 3.1-7-6 urn t h i c k , and i s smooth. I t i s e o s i n o p h i l i c . With the Periodic Acid S c h i f f reagent, an upper and a basal layer are revealed i n the tegument. The upper l a y e r ' i s approximately three timgg the thickness of the basal l a y e r . I t does not s t a i n b r i g h t l y with the S c h i f f reagent, which the lower layer does. The s t a i n i s diastase f a s t . The tegument of the ecsoma follows the contour of the ecsoma which proceeds a n t e r i o r l y f o r a short distance a f t e r meeting the soma. I t then makes a U-turn p o s t e r i o r l y following the length of the ecsoma. 3 3 The tegument of the ecsoma i s 5.4-10.8 urn t h i c k . The annulations seen i n whole mounts appear as blocks with a deep -V-shaped notch i n l o n g i t u d i n a l sections. The tegument i s e o s i n o p h i l i c due to i t s cytoplasmic nature. The PAS reagent d i f f e r e n t i a t e s the ecsomal tegument into three l a y e r s : an upper, a middle and a basal l a y e r . The upper and basal layer are equally t h i n - c u and s t a i n deeply with the S c h i f f colour. The middle layer i s approximately three times the thickness of the upper or basal layer and stains f a i n t l y with the S c h i f f s t a i n (Fig. 9). The whole tegument i s f a i n t l y diastase l a b i l e . B a l l o o n - l i k e i n f l a t i o n s on the tegument, observed i n whole mounts, were also v i s i b l e i n sectioned flukes ( F i g . 10). The-glassy i n f l a t i o n s appeared on the upper layer of the ecsomal tegument and did not s t a i n with haematoxylin. They were v i s i b l e i n specimens stained with PAS, but d i d not s t a i n . The i n f l a t i o n s did not appear along the en t i r e length of the ecsoma but were located i n parts here and there. The soma region showed none of these tegumental i n f l a t i o n s . : A t h i n fuzzy coat, absent on the tegument of the soma, was seen on the ecsomal tegument. The coat appeared granular but d i d not s t a i n with haematoxylin or eosin. I t disappeared i n sections stained f or carbohydrates. ii)''Muscuiatur-e: Beneath the tegument of the soma l i e s a.layer of c i r c u l a r and l o n g i t u d i n a l muscles. The two muscle•layers l i e i n close proximity and are. well developed. 34 The arrangement of the muscle layers i n the ecsoma i s i n the reverse order of that found i n the soma. Longitudinal muscles are followed i n depth by c i r c u l a r muscles. Both layers of muscle are . weak t(Fig.. l l ) . The r e t r a c t i o n and p r o t r a c t i o n of the ecsoma i s c a r r i e d out by sets of powerful protractor and r e t r a c t o r muscles (Fig. 12). These muscles are aggregated into several bundles and are d i s t r i b u t e d below a layer of peripheral parenchymal (mesenchymal) c e l l s . They continue into the soma and terminate anterior to the o r a l sucker. A few of these retractor, f i b r e s bent.'backwards where the-ecsoma. i s folded at the junction with the soma ...(Fig. 13). Longitudinal muscles also surround the d i g e s t i v e caeca i n the parenchyma. These are not aggregated into bundles, l i k e the r e t r a c t o r muscles, and are t h i c k single f i b r e s (-Fig. 14). i i i ) The Parenchyma: Parenchyma occupies ther_-pspace;-.beneath the muscle laye r s . It i s composed of fibrous and granular t i s s u e . In l o n g i t u d i n a l sections, the fibrous material i s seen to form a network of vacuolated spaces. The network appears f r a g i l e i n the soma because-it i s penetrated by reproductive organs. I t gradually becomes more compact as i t reaches- the p o s t e r i o r part of the ecsoma. The granular t i s s u e surrounds the digestive caeca y d and i s c e n t r a l l y l o c a t e d ' i n the ecsoma. The parenchyma stains with eosin and PAS. The PAS i s diastase l a b i l e i n d i c a t i n g the presence of glycogen. Embedded i n the periphery of the parenchymj.and arranged along the l o n g i t u d i n a l axis of the worm are three types of c e l l s . 35 Type 1 c e l l s 1 have a rounded nucleus with an eccentric endosome (Fi g . 15). The n u c l e i measure 3.7-5.0 um i n diameter. The n u c l e i a c t u a l l y belong to a syncytium of cells.. Several .nuclei are found surrounded by a mass of cytoplasm. Other n u c l e i do not appear to have cytoplasm around them, and l i e i n a f i n e network of the parenchyma. The n u c l e i do not s t a i n with eosin or PAS. Haematoxylin stains the endosome deeply. The n u c l e i are more abundant i n the ecsoma than i n the soma. Type 2 c e l l s (Fig. 16) are i r r e g u l a r l y shaped. The c e l l i s f i l l e d with cytoplasm and.has a c e n t r a l l y placed nucleus. Measurements taken for f i f t e e n c e l l s range from 12 x 8 ,um to l6 x 8 ;um, the average being 14.3 x 8 urn. The cytoplasm i s smooth, and stains with eosin. The PAS s t a i n i s diastase f a s t . The nucelus has a small endosome and stains with haematoxylin. The r a t i o of these c e l l s i n the soma and ecsoma i s 1:15. Type 3 c e l l s ( F ig. 17) are round with c e n t r a l l y placed, sph e r i c a l n u c l e i . The c e l l s measure 6.5-8.0 ;um i n diameter. The cytoplasm i s granular and stains with eosin, but not with PAS. The 2/um large nucleus stains with haematoxylin. There i s no endosome. These c e l l s are very few compared to the Type 1 c e l l s , and are found mostly i n the centre of the parenchyma, among the granular t i s s u e . " - '.. •'•<•' • i^tochemistry 36 f ) Enzyme histochemistry . Al k a l i n e phosphatase could' not be demonstrated i n the tegument of the soma or of the ecsoma. Acid phosphatase was l o c a l i z e d i n the ecsomal'tegument (Fig. 18). • In cryostat' and l o n g i t u d i n a l sections, i t could not be discerned whether t h i s l o c a l i z a t i o n was also shown by c e l l s i n the parenchyma. The c e l l u l a r • layer appeared black due to lead-.U- sulphide deposit, but t h i s could have'been due to a spreading of the deposit from the tegumental l a y e r . -In one serie s of s l i d e s , the phosphatase appeared c to be l i m i t e d to the upper layer of the tegument. No a c i d phosphatase was detected i n the control sections. The appearance of acid phosphatase on the tegument of the ecsoma was confirmed by incubating l i v e worms i n Gomori's mixture. Black lead sulphide was deposited i n the ecsoma tegument. This deposit was not, however, continuous f o r the ent i r e length of the ecsoma. I t appeared, .instead, i n patches randomly (Fig. 19). The phosphatase appeared to be concentrated at the l a t e r a l edge of the ecsoma. The phenomenon could be due to the ..displacement of the sulphide from the surface where the cover s l i p had pressured the ecsoma. In the l i v i n g worm, the phosphatase a c t i v i t y d i d not extend below the tegumental layer (Fig. 20) and appeared to be confined to the surface of the tegument. 37 g) Uptake of C glycine 14 The r e s u l t s of the C glycine uptake are, p l o t t e d i n F i g . 21. The experiment was repeated: twice. Exact d u p l i c a t i o n was impossible due to the presence of some unavoidable circumstances i n the experiments. These v a r i a b l e s were age and condition of the worms. In no case.were the worms a l l of the same age or siz e group. Worms were at times used af t e r they-had been l e f t i n the sa l i n e s a l t s o l u t i o n f o r two'or three days. Worms were also subjected to.pressures from handling during l i g a t i o n . However, the basic trend i n the two experiments.reflects a l i n e a r uptake of glycine. The low i n i t i a l N value indicates uptake was not a surface adsorption phenomenon. ' iese data, d e j T - j . ^ ' t i - 1 . e .the .' '" •• '"Le ecsoma tc; absorb g.lvo '.•«* . ~ _ • iflst; a couceutr&t2 ' . u d i f f e r e n c e " ..'- . ->£umex\ • , h) Tests f or p r o t e o l y t i c enzymes The areas exposed to the ecsomal extract on the black emulsion surface of the photographic f i l m appeared white, and therefore lysed. Similar white areas appeared on the film.exposed to 1% t r y p s i n . D i s t i l l e d water and marine t e l e o s t s a l i n e did not however produce such areas. The white areas were i n d i c a t i v e of the p r o t e o l y t i c a c t i v i t y of the ecsoma. Because the lysed areas on the f i l m were too f a i n t , the method was abandoned for the method below. In 15 tes t s using 6% g e l a t i n f i l m as the medium to be ly s e d , the procedure produced superior r e s u l t s . The ecsoma extracts lysed the g e l a t i n f i l m . Lysed regions appeared c l e a r , while the surrounding 38 g e l a t i n that did no ly s e stained blue with haematoxylin (Fig. 22). Control s l i d e s d i d not show any l y s i s . Lysed areas were produced around the ecsoma (Fig. 23) of worms that were l i g a t e d and incubated on a g e l a t i n f i l m base. There was no l y s i s i n the g e l a t i n around the somal region. 39 DISCUSSION- ;" -There i s no evidence from f i e l d observations to support Looss' (1907) hypothesis that the ecsoma i s protracted during favourable periods i n the stomach of the f i s h . Favourable periods were not defined by Looss, and i t could have implied a number of fa c t o r s . However, i n view.of h i s suggestion that the ecsoma withdrew during periods of a c i d i t y i n the stomach, i t was understood that by 'favourable periods' he meant a neutral or near-neutral pH value. Data show that despite the neutral 'andvhear-neutral pH values i n f i s h stomachs, parasites recovered from these did not have a protracted ecsoma. Looss (1907) suggested that the ecsoma was extended i n order to absorbs-nutrients. In the parasites observed i n the f i e l d and i n the laboratory, the ecsomas appeared mostly i n the ret r a c t e d p o s i t i o n . Factors responsible for the., protraction, of the ecsoma could not be established.. .Neither changes i n pH nor changes i n s a l i n i t y caused protraction.of the ecsoma. Although other factors such as redox p o t e n t i a l or the presence of various ions which are found i n the stomach of P l a t i c h t h y s s • f l e s u s (Mackenzie and Gibson, 1971) could influence the p r o t r a c t i o n , and r e t r a c t i o n , i t i s possible that the ecsoma could be extended by simple mechanical pressure. I t i s also possible that p r o t r a c t i o n could be caused by g a s t r i n found i n f i s h stomachs. 40 It i s here suggested that when food p a r t i c l e s are forced through the oesophagus, parasites are pressured against the oesophageal wall of the stomach, thereby p r o t r a c t i n g the ecsoma.. The e f f e c t would be s i m i l a r to pressuring, the worm with a cover s l i p against a s l i d e . Mechanical pressure would be greater or greatest close to the oesophagus where the stomach i s narrower. Hence, the worms would be expected to .be located here i f the ecsoma were to be protracted by the pressure of food entering through the oesophagus. Indeed, i t i s found that most of the T_;_ l i n d b e r g i found i n the stomach are 'located i n the cardiac region. This i s i n contrast to the observation of Macknezie and Gibson (1971) who found Hemiurus .communis, a t a i l e d hemiurid, i n the posterior part of the stomach of Platichthys f l e s u s . Although the p o s i t i o n i n g i n the two respective regions could be due to various other physico-chemical f a c t o r s , i t can be seen that the l o c a t i o n of 1L_ communis i s at a point where the stomach i s narrower and therefore subject to great pressures from food entering the i n t e s t i n e . In e f f e c t , the ecsoma of H. communis could also be extended by simple pressure i n order to absorb .food from the outside medium. The histochemistry of the ecsomal and the somal tegument suggests the tegumentsiStre glycoprotein or muc.oprotein -in composition. This i s i n conformity with the histochemical study of other trematodes reported i n the l i t e r a t u r e (see Lee 1966, 1972; Erasmus, 1972). According to Barka and Anderson (1965), mucoproteins are those compounds with a hexoseamine content higher than k%. With the S c h i f f 41 reagent, they give a strong PAS re a c t i o n . Glycoproteins with l e s s than" k% hexoseamine content s t a i n weakly with PAS'. On t h i s has i s , the upper tegumental layer on the soma i s a glycoprotein and the basal layer a mucoprotein. The upper ad and basal layers i n the ecsoma are .mucoproteins while the middle layer i s a glycoprotein. Since the teguments do not s t a i n with A l c i a n blue, there i s no acid mucopolysaccharide i n the tegument- Monne (1959) considered the a c i d mucopolysaccharide lajcer to protect the parasite against host enzymes. The function of a c i d mucopolysaccharide i n platyhelminth parasites i s . debatabl£e'='(-Rahemtulla and Lovtrup, 1974). In Tubulovesicula  l i n d b e r g i , which l i v e s i n a widely f l u c t u a t i n g chemical environment, c e r t a i n l y there appears no pro t e c t i o n by the acid mucopolysaccharide. It i s possible that protection i s afforded by a layer of glycans, which i s not v i s i b l e i n l i g h t microscope preparations. Such glycans, believed to function i n the pro t e c t i o n of worms, have been determined on the tegument of trematodes using e l e c t r o n microscopy (Lumsden, 1975)-(Similarities and diff e r e n c e s exist between the c e l l types and c e l l arrangement i n l i n d b e r g i and that i n Apoblema appendiculatum, Hemiurus C ' a a m n n j b , The. n u c l e i present i n the c o r t i c a l parenchyma of I\_ l i n d b e r g i belong to the s y n c y t i a l type of cell^'described by Pratt (1898), and Lander (1904). The submuscular c e l l s i n A. appendiculatum may be compared to the Type 2 c e l l s i n TV l i n d b e r g i ; the measurements do not compare, however.. While these submuscular c e l l s are only located beneath the tegumental muscle layers i n the soma of A^ appendiculatum } 42 the Type 2 c e l l s i n T\_ l i n d b e r g i are present i n the c o r t i c a l and medullary parenchyma i n the soma and ecsoma. As Lander (1904) c i t e s no measurements f o r the c e l l s i n IL_ crenatus, no comparisons can be made. A comparison between c e l l types i n T^ l i n d b e r g i and other trematodes shows some differences and s i m i l a r i t i e s . No s y n c y t i a l layer of c e l l s and n u c l e i were mentioned i n Gorgoderina attenuata, Loxogenes arcanum and Pneumonoeces breviplexus by Odlaug (1948), although he d i d describe subcuticular c e l l s with c e l l - w a l l s beneath the peripheral muscles i n the parenchyma for L. arcanum.'TThese were absent i n G^ attenuata and P^ breviplexus. In Lu_ arcanum some of the small subcuticular c e l l s were grouped together, with processes leading to the tegument. These groups of c e l l s may have been.syncytial, and therefore s i m i l a r to the Type 1 c e l l s observed i n T_^  l i n d b e r g i . The Type 1 c e l l s i n T^ l i n d b e r g i are also s i m i l a r to the ;sbeta-cells described i n Haem'atoleeehus'i cohfusus by Cheng and Provenza (i960). Odlaug (1948) described other c e l l types i n the parenchyma. None of these overlap the measurements i n Type 2 and Type 3 c e l l s found i n T. l i n d b e r g i . However, Type 3 c e l l s are s i m i l a r to the parenchymal c e l l shown i n the photographs for P^ breviplexus, and V' ..' t o s t h e a l p h a - c e l l s shown i n H.. cohfusus. . The c e l l s i n the parenchyma-of digenetic trematodes have been given d i f f e r e n t names by d i f f e r e n t authors. They are c a l l e d sub-muscular c e l l s (Pratt, 1898), subcuticular c e l l s (Lander, 1904),. 43 subcuticular and parenchymal c e l l s (Odlaug, 1948), and mesenchymal c e l l s by Hyman (±951). Some of these c e l l s are the tegumental c e l l s (also c a l l e d subtegumental c e l l s , perikarya, cytons) found i n electron microscope studies, and other., are parenchymal c e l l s . Speculation as to the function of these c e l l s i s diverse. They ( i ) contributed material to the surface of the tegument to protect the worm against host enzymes (Pratt, I898); ( i i ) formed muscle layers (Lander, 1904); ( i i i ) gave r i s e to lymphocytes and gonads (Hyman, 1951). E l e c t r o n micrographs show these c e l l s contribute to the tegumental l a y e r . Erasmus (1967c).found secretory bodies i n the c e l l s i n . Cyathocotyle bushiensis and he speculated these secreted the glycocalyx found on the surface of the tegument. TheTType; 3 c e l l s i n T. l i n d b e r g i are storage c e l l s f o r p r o t e i n , judgingby t h e i r l a r g e r s i z e and t h e i r diastase resistance to the Periodic Acid S c h i f f reagent. The parenchyma of the ecsoma contains much glycogen which i s probably used as an energy source f o r the p r o t r a c t i o n and r e t r a c t i o n of the ecsoma. The presence of a c i d phosphatase on the tegument of the ecsoma, and the absence on the soma, demonstrates that the ecsomal tegument i s - m e t a b o l i c a l l y a c t i v e . Phosphat ases are widespread i n nature and are - d i v i d e d / - i n t o , a c i d or a l k a l i n e depending on whether t h e i r optimal r e a c t i o n i s at an a c i d or a l k a l i n e pH. The stomach of the f l a t f i s h being a c i d i c , i t might be expected that the phosphatase on the parasite would be a c i d i c . A c id phosphatases have been detected on the teguments of F a s c i o l a hepatica, Schistosoma mansoni, Opisthioglyphae ranae (Halton, 196T) among the trematodes; thus the tegument i n the ecsoma i s s i m i l a r to the o v e r - a l l teguments of these trematodes, and i t i s the soma which i s d i f f e r e n t . Phosphatases are, however, un i v e r s a l among helminth teguments'. The r o l e of phosphatases i s speculative. Read (1966) c r i t i c i s e d the older theory that phosphatases phosphorylated hexose sugars during, transport. He postulated that the enzymes were r e l a t e d to metabolic c o n t r o l or the modif i c a t i o n of solutes to which c e l l s were otherwise impermeable. Lumsden et a l . (1968) and Threadgold (1968) suggested that they might hydrolyse phosphate esters at the plasma membrane to f a c i l i t a t e transport through the membrane. Erasmus (1972) i s emphatic that phosphatases play a -role i n act i v e transport. Although phosphatases may have a broad s p e c i f i c i t y (de Duve, 1969)9 most phosphatases are r e l a t e d to the process of dige s t i o n ( P i t t , 1975). Food may be taken i n by pinocytosis and digested by the acid 1 hydrolases contained i n lysosomes. or .microsomes., A l t e r n a t e l y , lysosomes may secrete a c i d phosphatase ad and digest food by exocytosis (Smith and Farquhar, 1966). The presence of a c i d phosphatase ont' the tegument of the ecsoma suggests that the tegument c a r r i e s out dige s t i v e processes. The uptake of rad i o a c t i v e glycine through the tegument of T. l i n d b e r g i can be compared with the studies c a r r i e d out by Mansour (1959), I s s e r o f f and,Read (1969), Nollen (1968a, b) and Asch and Read (1975). These authors demonstrated that hexose sugars and amino acids could be transported through the tegument of various p a r a s i t e s . 45 In the present study i t i s evident that the ecsomal tegument of T. l i n d b e r g i allows an accumulation of glycine against a concentration gradient when the ecsoma i s l i g a t e d a n t e r i o r l y and p o s t e r i o r l y . The study leads to the speculation that the ecsoma i s capable of absorbing other metabolites such as amino acids from i t s surrounding medium,r""d .the -pi >cess '">y c t i -e transport. The s e c r e t i o n of p r o t e o l y t i c enzymes have been demonstrated i n F. hepatica ( T h o r s e l l and Bjorkman, 1965) » C;. .bushiensis (Erasmus and Ohman, 1963)9 Cyathocotyle and Holostephanus spp. (Erasmus, 1972). P r o t e o l y t i c enzymes are capable of degrading protein substrates and have been described i n extracts of other parasites. The p r o t e o l y t i c a c t i v i t y of parasites have been assigned to penetrating skins, as i n S. mansoni cercariae (Lee and. Lewert, 1957). Among other p a r a s i t e s , Erasmus (1972) proposed an 'extra-corporeal d i g e s t i o n " function f o r the p r o t e o l y t i c enzymes. According to the 'extracorporeal digestion' theory, the secretion of enzymes by the para s i t e lyses the host material around the p a r a s i t e . This lysed material i s then ingested. Erasmus and Ohman (1963) reported l y t i c enzymes i n the adhesive organs of bushiensis. The authors further showed that the adhesive organs took up nutrients that were lysed by the enzymes. The 'extracorporeal d i g e s t i o n ' theory' i s no d i f f e r e n t from the membrane or contact d i g e s t i o n theory advocated by Ugolev (1968). Ugolev proposed that membrane di g e s t i o n occurs on the i n t e s t i n e of vertebrates. Enzymes present on c e l l surfaces are responsible f o r 46 digestion of e x t r a - c e l l u l a r m a t e r i a l . Smyth (1972) presented evidence that membrane digestion occurred i n Moniezia expansa. The dige s t i o n appears to have occurred a f t e r the secretion .of substances from within the tegument. The b a l l o o n ^ l i k e i n f l a t i o n s seen on the tegument of the ecsoma may be apocrine secretions associated with p r o t e o l y t i c activity.-The.results of the experiments and the discussion i n the foregoing paragraphs make i t clear that the ecs.oma i s a p h y s i o l o g i c a l l y a c t i v e organ. I t consists of a parenchyma which is. capable of storing glycogen. Some of the c e l l s within.the parenchyma store p r o t e i n ; others probably produce the muscle layers and the tegument. The tegument i s i t s e l f m e t a b o l i c a l l y dynamic and i s capabeke of secreting enzymes which.are p r o t e o l y t i c . These enzymes probably break down proteins into amino acids and- these are transported through the tegument into the c e l l s within. It i s thus seen that.the ecsoma i s capable of carrying out 'extracorporeal digestion' as enunciated by Erasmus (1972) or 'membrane-digestion' as advocated by Ugolev (1968) for vertebrate c e l l s . 47 SUMMARY 1. This work demonstrates that the tegument on the soma .consists of an outer glycoprotein and an inner mucoprotein. The tegument cm the ecsoma i s a t r i - l a y e r ; the outer, middle, and inner layers : •-• are mucoprotein, glycoprotein and mucoprotein:, .respectively. 2. The tegument on Tubulovesicula l i n d b e r g i does not coniainsacid mucopolysaccharides, and i s therefore d i f f e r e n t from the tegument of a majority of trematodes which have a c i d mucopolysaccharides. 3. The ecsoma shows acid phosphatase a c t i v i t y , which the soma does not. I t i s therefore believed t h a t the ecsoma i s me t a b o l i c a l l y a c t i v e , and absorbs food through i t s tegument. 4. The ecsoma i s capable of taking up glycine through i t s tegument. It i s presumed that other metabolites may also be transported through the tegument of the ecsoma. 5. The, ecsoma produces p r o t e o l y t i c enzymes. It i s suggested that these break down proteins and peptides to amino acids which are then transported across the ecsoma tegument. 6. Since the worm i s found with i t s ecsoma retracted" and i s located -mostly at the cardiac region of the stomach, i t i s possible that the ecsoma i s protracted by te: the pressure of food against the parasite as i t i s swallowed by the f i s h host. 48 TABLE la Incidence of infestation and incidence of infection of flatfish by Tubulovesicula lindbergi in Georgia Strait Area of Fish species  catch 1. Unknown -fish supplied by MacMillan Fisheries fish # arid * of examined fish parasitized # parasites/fish Eopsetta jordani Lepidopsetta  bilineata Parophrys  vetulus Hippoglossus  stenolepis Microstomus  pacificus Crescent Beach £». vetulus L. bilineata Georgia Strait - no fixed locality; fish supplied by Pacific Biol. Stn, Nanaimo 5 (62.5*) "2 .(60.0*) 3 (75*) 1 (100*) 3.66 6.50 8.6 h P. vetulus 1 (25*) 25 Hippoglossoides  elassodon 11 2 (18.18*) 2 Area of catch Fish species -' U. English Bay P. vetulus Psettichthys  coenosus Pleuronichthys  melanostictus Lepidopsetta  bilineata Isopsetta  isolepis Platichthys  stellatus Hippoglossoides  elassodon 5- Little Campbell River P. stellatus 6. North Army Jetty P. stellatus P. vetulus L. bilineata P. melanostictus 7. Port Moody P. melanostictus 8. White Hock P. vetulus L. bilineata 4 -fish # and % of  examined fish parasitized 37 1* (10%) 6 U (66.6%) 6 6 (100%) 12 1 (8.33?) 1 1 (100?) 28 9 6 1.(1.6650' 69 9 (13.OW 97 h (k.12%) 12 2 (16.6?) 11 20 h 3 (15%) 1 1 (100?) # parasites/fish 6.5 6.1 6 3 2 2 6.7 5-75 5. 8.6 50 Area of catch F i s h species 9. Squamish Estuary P. s t e l l a t u s 1 0 . Stewert Channel Reinhardtius  hippoglo s so i de s P. vetulus P. s t e l l a t u s L. b i l i n e a t a P. melanostictus M. p a c i f i c u s # f i s h # and * of  examined f i s h p a r a s i t i z e d § p a r a s i t e s / f i s h 1*3 1 1 1 1 1 1 0 1 0 1 6 (37.20*) 1 . 2 1 ( 1 0 0 * ) 1 2 ( 1 8 * ) 1 TABLE l b . SUMMARY OF T. l i n d b e r g i INFECTIONS F i s h species # f i s h examined # f i s h in fec ted * in fect : P. coenosus 6 1* 66.0 R. hippoglossoides 1*3 16 37.2 L. b i l i n e a t a 29 7 2U.13 P. melanostictus 38 6 15.75 P. s t e l l a t u s 110 12 lo.90 H. elassodon 20 2 10.0 P. vetulus 166 15 9.05 M . p a c i f i c u s 13 - -H. s tenolep is 3 - -51 TABLE I l a . Variations i n pH i n f l a t f i s h stomach  and condition of ecsoma PH F i s h species examined # f i s h examined Stomach Empty-Retracted Protracted Stomach Retracted F u l l Protracted 3 M. p a c i f i c u s L. b i l i n e a t a R. hippoKlossoides 1 1 1 1+ Glyptoc ephalus zachirus R. hippoRlossoides 1 2 1 5 P. vetulus 2 R. hippoRlossoides 3 1 1 6 R. hippoRlossoides L". b i l i n e a t a P. vetulus P. s t e l l a t u s 7 2 1 1 3 1 R. hippoRlossoides P. melanostictus 8 1 11 3 Tota l 30 k 13 4 TABLE l i b . P o s i t i o n of T. l i n d b e r g i in  stomach of f i s h examined i n  the f i e l d Cardiac sect ion 15 Middle sec t ion 6 P y l o r i c sec t ion 0 TABLE H e . Or ienta t ion of T. l i n d b e r g i mouth i n the stomach of f i s h A n t e r i o r l y d i rec ted l l * L a t e r a l l y d i rec ted 3 P o s t e r i o r l y d i rec ted 1* TABLE l i d . P o s i t i o n o f T. l i n d b e r g i i n  stomach of f i s h examined i n  the laboratory Cardiac sect ion 17 Middle sect ion 6 P y l o r i c s e c t i o n 1 5 TABLE l i e . Or ienta t ion of T. l i ndberg i  mouth in the stomach of f i s h A n t e r i o r l y d i rec ted L a t e r a l l y d i rec ted P o s t e r i o r l y d i rec ted 21 2 1 TABLE III. Effect of pH on the ecsoma of  T. lindbergi The ecsoma responded by protracting (+) or remaining retracted (-). The period it remaind protracted or retracted is indicated in column (d). The survival period of the parasite is shovn in (e). (a) PH (b) of worms (c) Ecsoma  retracted/  protracted (d) Time retracted/  protracted (e) Time parasite survived 30 minutes 60 30 minutes 60 6 7 8 9 10 11 1 2 1 1 3 3 1 2 2 2 1 1 120 " 2k hours 2k hours 210 minutes 2k hours 2k0 minutes 2k hours 2k hours 2k hours 3 hours 2k hours 2k hours 2k hours 150 minutes 2k hours 120 2k hours 2k hours 2k hours 2k hours 2U0 minutes 2k hours 2k hours 2k hours 2k hours 2k hours 2k hours 2k hours 150 minutes 2k hours 54 TABLE I V . . E f f e c t of s a l i n i t y on the p ro t rac t ion  of the ecsoma and the surv iva l of  T . l i n d b e r g i The ecsoma responded by protracting(+) or remaining r e t r a c t e d ( - ) . The per iod i t remained protracted i s ind ica ted i n column (d) . The s u r v i v a l per iod of the paras i te i s shown i n column (e) . (a) '(*) (c) (d) (e) Degree of s a l i n i t y (c f . marine te leos t s a l i n e (0.78*)) § paras i tes Pro t rac t ion +1-Pro t rac t ion per iod Time s u r v i v a l 2X 3 - 19 hours 1.8X 3 18 hours 1.6X 3 - 22 hours 1 + 1 hour 2k hours 1.1*X 3 - • 23 hours 1.2X 3 - -. 2k hours 1.0X 1* 2k hours 0.8X 3 ' 1 + 7 hours 2k 2k hours hours 0.6X 2 1 + 9 hours 2k 2k hours hours o.i»x 2 1 + 23 hours 2k 2k hours hours 0.2X 2 1 + 23 hours 2k 2k hours hours D i s t i l l e d water 5 — 5 hours 55-56 . v a l l e y p i n o c y t o t i c v e s i c l e t e g u m e n t c i r c u l a r m u s c l e — l o n g i t u d i n a l m u s c l e t e g u m e n t a l c e l l p a r e n c h y m a l c e l l n u c l e u s 2 The u l t r a s t r u c t u r e of.the tegument • "•' of F a s c i o l a hepatiea. . (Redrawn af t e r Threadgol'd, 1963) 57 oral sucker pars prostatica ventral sucker seminal vesicle testes .ovary v i te l lar ia D M A E C S O M A uterus caecum excretory bladder Tubulovesicula lindbergi 24 21 18H 1 2 - J 9 H 6 J 3 J 0-2x l.Ox 1.4x l.&x 2.Ox d i s t i l l e d w a t e r T e l e o s t sa l ine concentration Fig. 4 Graph shoving a b i l i t y of T. l i n d b e r g i to survive i n varying degrees of s a l i n i t y . -Figure represents worms and. the means of times of the p a r t i c u l a r concentration of saline indicated i n Table IV. \ 59 Figure 5. Figure 6. Figure 7. "B a l l o o n - l i k e " i n f l a t i o n s are seen on the tegument of the ecsoma i n a whole mount of l i n d b e r g i . Formalin f i x e d . Carmine stained. 450X. Extra-tegumental coat v i s i b l e on the ecsoma i n a whole mount. Formalin f i x e d . Harris' haematoxylin s t a i n . O i l immersion. Ecsoma-soma junction. Where somal tegument meets ecsomal tegument, a c l e f t i s seen. Haematoxylin-eosin s t a i n . 100X. 59*. 6o Figure 8. L.S. Tegument of the soma. Note M - l a y e r . FAS s t a i n . L 5 O X . ' • * Figure 9- L.S. Tegument of the ecsoma. Note t r i - l a y e r PAS s t a i n . 450X. Figure 10. "B a l l o o n - l i k e " i n f l a t i o n s on ecsomal tegument. L.S. PAS s t a i n . O i l immersion. 60 CL 10 61 L.S. Ecsoma with l o n g i t u d i n a l and c i r c u l a r muscles "beneath tegument. Arrow marks l o n g i -t u d i n a l muscle f i b r e s . H &E. O i l immersion. T.S. Ecsoma with r e t r a c t o r muscles i n parenchyma. H & E. O i l immersion. L.S... Retractor muscles i n soma and ecsoma. Muscles from ecsoma. go into soma; others Lend around wher.e~ ecsomatturns to.-proceed and meet soma. T.S. Retractor, muscles i n single f i b r e s around caecum. H & E. O i l immersion. 6itx 62 Figure 15. Type 1 c e l l i n parenchyma below tegument i n ecsoma- Note s y n c y t i a l nature: n u c l e i with endosome. H & E. O i l immersion. Figure 16. Type 2 c e l l i n parenchyma of ecsoma. Note large c e n t r a l nucleus. H & E. O i l immersion. Figure IT. Type 3 c e l l i n parenchyma of ecsoma. beneath tegument. 62 a 63 Figure 18. L.S. Acid phosphatase l o c a l i z e d i n and beneath tegument of ecsoma. Dark structures beneath tegument are presumably c e l l s . O i l immersion. Figure 19. L.S. a c i d phosphatase l o c a l i z e d on surface of ecsomal tegument. O i l immersion. Figure 20. W.M. Ecsoma with acid phosphatase. Phosphatase l o c a l i z e d on periphery of ecsoma due to pressure from cover s l i p . 450X. FIG.WI UPTAKE OF C 1 4 GLYCINE IN T. LINDBERGI ECSOMA 65 Figure 22 L y t i c a c t i v i t y of ecsomal homogenate on g e l a t i n f i l m . The lysed area i s seen as the clear halo around c e n t r a l spot. Figure 23. L y t i c area around ecsoma i s indicated by white. 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