HISTOCHEMICAL AN BIOCHEMICAD L STUDIES ON SOME … · histochemical an biochemicad l studies on...

HISTOCHEMICAL A N D BIOCHEMICAL STUDIES ON SOME

AMPHISTOME PARASITES A N D THEIR HOST

THESIS SUBMITTED FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY IN

ZOOLOGY

BY

SHAH MAQBOOL ALAM

SECTION OF PARASITOLOGY DEPARTMENT OF ZOOLOGY

ALIGARH MUSLIM UNIVERSITY ALIGARH (INDIA) DECEMBER, 1984

V v

T2911

{University : 285

Public : 6646

Sections

1 ENTOMOLOGY 2 PARASITOLOGY 3 ICHTHYOLOGY & FISHERIES 4 AGRICULTURAL NEMATOLOGY 5 GENETICS

D E P A R T M E N T OF Z O O L O G Y ALIGARH MUSLIM UNIVERSITY

ALIGARH, U. P. INDIA 202001 Ref No.

Oate Dec.20, 1984

This i s to c e r t i f y that the thesis e n t i t l e d

"Histochemical and biochemical studies on some

amphistome parasites and the i r host " , which i s being

submitted by Mr. Shah Maqbool Alam, embodies o r ig ina l

work done by the candidate himsel f . The ent ire work

was carr ied out under our supervision and that we allow

him to submit the same in fu l f i lment o f the requirements

f o r the degree o f Doctor o f Philosophy in Zoology o f

t h i s univers i ty .

i V ^ ff. Ather H. Siddiqi Professor Supervisor

Dr. Wajih A. Nizami Co-supervisor

CONTENTS

ACKNOWLEDGEMENTS

LIST OF ABBREVIATIONS . .

INTRODUCTION AND STATEMENT OF PROBLEM . ,

HISTORICAL REVIEW

MATERIALS AND METHODS .,

RESULTS AND DISCUSSION.,

SUMMARY AND CONCLUSIONS

APPENDIX A

REFERENCES

PUBLICATIONS

1

i i

1

9

29

41

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130

137

t u *

ACKNOWLEDC MENTS

I have great honour in expressing my sincere grate fu l -ness to my supervisor Professor Ather H. Siddiqi f o r his inspiring guidance and f o r his c r i t i c a l and useful observa-t ions while going through the manuscript. I am also thankful to my Co-supervisor Dr. Wajih A. Nizami f o r his keen interest during the course o f th is study.

Sincere thanks are also due to Prof . Nawab H. Khan, Chairman, Deptt. o f Zoology f o r providing necessary labora-tory f a c i l i t i e s .

I would l ike to thank my research col leagues, part i cu-l a r l y Mr. Jawed Siddiqui f o r helpful suggestions and Dr. Irfan Ahmad f o r assistance in photography of ge ls . I am indebted to Dr.(Mrs.) Kusura Saxena, Chairperson, Deptt. o f Gynaecology & Obstetrics , J.N. Medical College, A.M.U., f o r allowing me the use of the cryostat of her department, and to Mr. Mohammad Ashfaq Khan, Senior Technician in the same department f o r o f f e r i n g his services whenever required. Thanks are also due to Mr. Syed Mujeer f o r preparation of the microphotographic p lates . I appreciate the e f f o r t s o f Mr. Syed Shakir A l i , Lab. attendant, f o r regularly providing f resh material on request.

I acknowledge with gratitude the patience shown by my wife Dr. Rafia Parveen and the tremendous moral support provided by her during the preparation of th is thes i s .

Lastly, the f inancia l assistance provided by the Council o f S c i e n t i f i c and Industrial Research, New Delhi, i s grate-f u l l y acknowledged.

3hah Maqbool Alam

i i

LIST OF ABBREVIATIONS

PAGE - Polyacrylamide disc gel e lectrophoresis SDS - Sodium dodecyl sulphate AFA - Acetic formalin alcohol BPB - Bromophenol blue TRIS - Tris (hydroxymethyl) aminomethane TEMED - N,N,N,N-tetramethylethylenediamine BIS - N,N-methylene b is acrylamide CBB - Coomassie b r i l l i a n t blue NAD - Nicotinamide adenine dinucleotide NADP - Nicotinamide adenine dinucleotide phosphate Nitro BT - Nitroblue tetrazolium PMS - Phenazine methosulphate NaCN - Sodium cyanide G-6-PDH - glucose-6-phosphate dehydrogenase 6-PGDH - 6-phosphogluconate dehydrogenase MDH - Malate dehydrogenase SDH - Succinate dehydrogenase A1 DH - Alcohol dehydrogenase H-6-DH - Hexose-6-dehydrogenase

°<"-GPDH - <-glycerophosphate dehydrogenase G-3-PDH - Glyceraldehyde-3-phosphate dehydrogenase NDH - Nothing' dehydrogenase GPI - Glucose phosphate isomerase Fr1,6-DP - Fructose 1,6-diphosphatase PGM - Phosphoglucomutase HK - Hexokinase

I N T R O D U C T I O N

A N D

S T A T E M E N T OF P R O B L E M

The trematodes are knovai to i n f e c t a wide spectrxam

o f hosts and cause considerable damage to both p o i k i l o -

thermic as well as homoiothermic animals, hardly sparing

any organ systems. The siarvival of the parasites i s i n f l u -

enced by the general b i o t i c factors associated with the the

micro- and macro environments as well as by the intimate

physio log ica l and immunological interactions between the

parasite and the host, which forms the basis o f the host -

parasite relationship.

Our knowledge in the f i e l d o f parasite biochemistry

i s increasing day by day. The parasite biochemists are

employing latest experimental techniques which provide

increasingly more accurate results . However, most o f the

studies have been restr i c ted to some model species l ike

Schistosoma mansoni and Fasciola hepatica among trematodes

^^^ Ascaris lumbricoides representing the nematodes. Many

other equally important species o f trematodes from the point

o f view of their veterinary importance, have remained more

or l e s s neglected. Also very rarely a biochemical study i s

accompanied or supplemented by a histochemical f inding and

v i ce versa.

Most of the f indings of biochemical and physiological

nature are based on to ta l worm homogenates because the i s o -

la t i on of the d i f f e rent organs and organ systems i s imposs-

i b l e , The results thus obtained may not provide possible

c lues to the b i o l o g i c a l s ignif icance o f a particular subs-

tance, in relat ion to s p e c i f i c organs and organ systems.

To overcome th is d i f f i c u l t y to some extent, the b ioche-

mical and physiological studies should be supplemented by

histochemical studies. Recent advances in the f i e l d of

histochemistry have made this task poss ib le . Spec i f i c

staining techniques give exact l o ca l i za t i on and intensity ,

thus revealing the degree of involvement of the substance(s)

in a biochemical reaction in that region.

The role of enzymes in regulating various l i f e pro -

cesses in an organism i s very well known. Similarly t r e -

matodes are also reported to have various enzymes and

enzyme systems. The enzymes so far studied electropho-

r e t i c a l l y in most cases are confined to acid and alkaline

phosphatases, general esterases and l a c t i c dehydrogenase.

Other equally important enzymes which play key ro les in

d i f f e r e n t metabolic processes have remained some what

neglected. Interestingly the reports are based either on

histochemical observations or purely on biochemical e s t i -

mations. In the former case only the location of a c t i v i ty

i s detected while in the la t ter only the quantity of enzy-

me ( s ) becomes known. Hence there ex is ts an urgent need

to study the d i f f e rent enzymes and enzyme systems, h i s t o -

chemically as well as e lectrophoret ical ly in order to

observe the exact locat ion of the enzyme and the number

and pattern of the isoenzymes respect ively . I f the i s o -

enzymes exist then their possible fxinction in the biochemi-

c a l adaptation can be worked out. More interest ing results

can be obtained regarding the host-parasite relat ionship,

i f the parasite and the host t issue are examined simul-

taneously f o r a part icular study.

Some four decades ago an enzyme was regarded as a

s ingle entity . I t was seriously questioned later by

several workers a f t e r they foxind that an enzyme derived

from one source d i f f e r e d in several respects from that

obtained from another source. This led to spectacular

development of interest in the occurrence of enzymes in

multiple molecular forms, known as isoenz3niies or isoz3nnes

(Webb,1964).

The development of more refined techniques having

high resolution power f o r protein separation has in i t ia ted

interest in biochemical systematics and host-parasite

re lat ionship . Electrophoresis and i s o e l e c t r i c focussing

has been applied more widely f o r the separation of the

enzyme forms and other soluble proteins. Ployacrylamide

d i s c gel electrophoresis (PAGE) has also been found to be

more advantageous than other techniques because of short

rionning time and transparent ge l . This helps in greater

resolution and quick reproduction of results . The e l e c t r o -

phoretic studies are thus necessary to ascertain the num-

ber of molecular forms (isoenzymes)of an enzyme, p a r t i c i -

pating in a physiological process.

An "isoenzyme band" or simply "band" i s an e l e c t r o -

phoret i ca l ly d i s t inc t ive and active component of an enzyme.

Several such bands can occur in one cloned organism. The

one or several bands of a particular enzyme consistently

found in an organism i s the "isoenzyme pattern". As

Godfrey (l982) described, "The isoenzyme p r o f i l e i s a l l the

enzyme patterns discovered in the organism, i f the p r o f i l e

i s ' ident ical* between the organisms, these are said to be

s imilar .

The structural pecular i t ies that separate one taxo-

nomic entity from another may be observable only at the

microscopical l e v e l . Simple structural or morphological

characters are perhaps not authentic in identi fying orga-

nisms without considering their chemical composition, b i o -

chemical behaviour and changes induced in their respective

host organism as a result of in fec t i on .

The use of in tr ins i c characters that can be ident i -

f i e d by biochemical means has been named "biochemical

taxonomy". The term •molecular taxonomy' embraces both

the intr ins i c characters and immunological part icularly

sero log ica l characters. As we know that the same enzjrme

from the parasite and the host may catalyse same reaction

in both without being ident ica l , they may also be immuno-

l o g i c a l l y d i f f erent with d i f ferent physico-chemical

propert ies .

Taxonomic characterization of amphistomes in gene-

ral i s unclear. There arose controversies regarding the

v a l i d i t y of certain species because most of the taxonomic

studies were exclusively based on morphological characters

while other fac tors which influence the phenomenon of

parasitism were completely neglected, Sey (l979) exten-

s ive ly examined the va l id i ty and systematic posit ion of

certain paramphistomids of Indian ruminants and s3mony—

mized the fol lowing species : Paramphistomum epiclitum

(Fischoeder,1904) S3m: Cotylophoron indicum (St i l e s and

Goldberger,1910); P. thapari (Price and Mcintosh,1953);

Srivastavia indica (Singh,1970) on the basis of the

pattern of muscle bundles. Therefore, i t was decided

that some biochemical parameters l ike isoenzyme p r o f i l e

should be studied, ' The present study on the isoenzymes

of the three amphistomes of buf falo i s to examine the

relat ionship of these trematodes with each other and to

invest igate the influence of the habitat on the i s o -

enzyme patterns of the parasites in the same host i f any.

The economic importance of c a t t l e spec ial ly in

India as well as throughout the world i s quite well known.

In th is part of India they are always heavily infected

with trematodes. Of the prevalent in fec t ions , the most

common ones are Gastrothvlax crumenifer. Paramphistomum

epicl itum. and Gigantocotvle explanatum. Consequently

the health of the host ( ca t t l e ) i s adversely a f f e c t e d

and the animal becomes succeptible to many other secon-

dary microbial in fec t ions . This further deteriorates the

condition of the animal and therefore , causes considerable

l o s s to our animal based industry. In view of these fa c t s

the present study w i l l provide the basic knowledge of

metabolic processes as well as host-parasite r e l a t i o n -

ship. Such studies on parasites and the ir respective

host t issues , may become subservient to chemotherapeutic

aspects as well as give impetus to the studies on b i o -

chemical taxonomy.

In the present Investigation an attempt has been

made to study the various components l i k e amyloids, acid

mucopolysaccharides, col lagen, e las t in , f i b r i n , tr j^ito-

phan and nucleic acids (DNA and RNA) and the fo l lowing

enzymes (mainly involved in the carbohydrate metabolism

and related pathways), on a comparative basis in a l l the

three species of amphistomes, inhabiting two d i f f e rent

habitats in the same host ( bu f fa l o ) . The enzymes include

glucose-6-phosphate dehydrogenase, 6-phosphogluconate, malate,

succinate, alcohol , -K-glycerophosphate, glyceraldehyde-3-phos-

phate, hexose-5- and ^nothing' dehydrogenases and glucose

phosphate isomerase, phosphoglucomutase, fructose 1,6-diphos-

phatase, hexokinase and aldolase. The electrophoretic obser -

vations on the enzymes of the parasites and the in fected host

t issues have also been made to f ind out the number and pattern

o f isoenzymes of each enzyme. This, probably, i s one of the

few reports o f i t s kind in the f i e l d o f parasite biochemistry

and physiology in which histochemical l oca l i zat ion has been

supplemented by electrophoretic studies so as to have a c lear

and conclusive understanding of the various enzymes involved.

The histochemical observations in th is study were made

by l i ght microscopy due to the lack o f a transmission electron

microscope in th i s department. Necessary controls were run

simultaneously f o r the appropriate interpretation of the

resu l t s .

As more and more fac t s are becoming known about the

biochemistry and physiology of trematodes, ever increasing

e f f o r t s are being made to develop the anthelmintics by

rationale approach rather than empirical approach. This can

only be achieved i f we know more and more about the nature

and properties o f enzymes and enzyme systems of the parasite

as well as of their host

8

It i s hoped that the results of th i s study w i l l prove

to be useful as a pos i t ive contribution in the vast f i e l d

o f parasite biochemistry and physiology. This should also

help others in carrying out more purposeful studies with

ever increasing new ideas and new techniques.

H I S T O R I C A L R E V I E W

In spite of many advances that have been made in the

f i e l d of trematode biochemistry and physiology in the last

few decades, there ex is t many gaps and lacunae in our know-

ledge . However, the histochemical studies on the compo-

s i t i o n and d i f f e r e n t i a l distr ibution of certain biochemical

substances in trematodes have been reviewed extensively by

von Brand ( l979) , Chappell ( l980) , Barrett ( l 9 8 l ) , and

Smyth and Halton (1983). Almost a l l the previous reports

have been in agreement that glycogen i s the most common

stored polysaccharide, evenly distributed in higher quan-

t i t y throughout the parenchyma of the trematodes.

The acid mucopolysaccharides have been reported from

the cyst wall of metacercariae; tegument, acetabular glands,

gut epithelium and the l ining of the excretory canal in a

number of trematode species by several workers (Monne,l959;

Stirewalt,1959; Dixon and Mercer,1964; Dixon,1965; Cheng

and Burton,1966; Rees,l967; Smith et al^l969; Porter,1970;

Nacheva,1975 ; Mol f i , l976 ; Nacheva,1977; Sharma and

Mandawat,1979 and Alam and Nizami,1984)o The functional

r o l e of these mucopolysaccharides has been suggested as

protect ion of the worm from the h o s t ' s harmful secret ions.

10

The amyloids have been described in chemical terms

as combinations of proteins with acid mucopolysaccharides

and are usually seen as massive deposits , depending on the

duration of disease, part icularly in the inflammatory d i s e -

ases . They are further c l a s s i f i e d as primary and secondary

on the basis of staining reaction d i f ferences . Amyloids

are microscopically homogenous, translucent, ac idophi l i c

substances which usually occur as extracel lular deposits ,

Alam and Nizami (l984) reported f o r the f i r s t time the pre -

sence of moderate amounts of amyloids in the parenchyma,

v i t e l l a r i a , gonads, musculature, suckers and uterus of the

metacercaria of Clinostomum complanatum.

The nucleic acids (DNA and RNA) have been known to

occur widely among helminths. A number of trematode spe-

c i e s have already been histochemically examined f o r the

l o ca l i za t i on of DNA and RNA (see l i terature von Brand,1979;

Chappell,1980; Barrett,1981; Smyth and Halton,1983). Great

variat ion in the amount of DNA in relat ion to the develop-

ment of v i te l lar ian c e l l s of F. hepatica was reported by

Govaert ( l953) . DNA was also l o ca l i zed by Murakami ( l96 l )

in the nuclei of mid gut ep i the l ia l c e l l s , the parenchyma-

tous c e l l s of the l a te ra l l ine , spermatogonia, spermatocytes

and spermatozoa and RNA from the basal tissue of the latera l

l i n e , the ep i the l ia l c e l l s of the mid gut, the cephalic and

cerv i ca l glands, the seminal receptacle , the uterus and the

developing germ c e l l s in the testes of C. bushinensis.

11

Further the s i t es of active protein synthesis were suggested

to have higher amounts of RNA (Erasmus and 6hman,l963).

Reznik (l963) and Gupta and Kapoor (l979) also reported wide

distr ibution of nucleic acids in_F. hepatica and C. bushinen-

s i s respect ively , while Alam and Nizami (l984) reported in te -

nse presence of RNA in the suckers and DNA in testes with

some moderate amounts in the ovary of the metacercaria of

C. complanatum. I t seems that in spite of the a'bove studies,

very l i t t l e i s known about the process of synthesis of nucleic

acids in trematodes.

The histochemical l oca l izat ion and d i f f e r e n t i a l d i s t r i -

bution of col lagen, e last in , f i b r i n and tryptophan in trema-

todes has been carried out f o r the f i r s t time in the present

investigation and no other report on the presence o f the above

substances i s avai lable ,

Histoenzymological studies:

Over the l as t many years, part icular ly in the las t

decade, numerous studies on the histochemical l o ca l i za t i on of

enzymes in trematodes have been carried out. The available

l i t e rature has been reviewed by von Brand (l979) and Smyth

and Halton ( l983) . Only acid and alkaline phosphatases and

general esterases (the two enzyme groups) have been studied

in d e t a i l . Otherwise such studies on other enzymes and

enzyme systems are scanty.

The previous reports by Dusanic ( l959) ; Robinson ( l96 l ) ;

Saito (1961); Ohman ( l965, l966) ; Bogitsh (1966a,1975);

12

Halton (1967a); Threadgold ( l968) ; Erasmus,1968,1970);

Bogitsh and Shannon ( l 9 7 l ) ; Probert ^ ( l972) ; Allen

(1972); Probert and Lwin ( l974) ; Rodgi ^ ( l976) ;

Sharma (1976) and Haque and Siddiqi ( l982) about the l o c a l -

izat ion of phosphatases (acid and alkal ine) in d i f f erent

species inhabiting d i f f erent habitats revealed that these

enzymes were almost ubiquitous in the i r distr ibution in

the trematode body organization. But in the case of

Hasst i l i s ia ov is . Soboleva and Zdarska (l983) reported that

except the microv i l l i the other t issues and organs did not

show posi t ive reaction f o r alkaline phosphatase while

Halton (l967a) reported the absence of acid phosphatase

a c t i v i t y in seven species of monogenea.

Most of the authors have been in agreement that the

phosphatases (acid and alkaline) are concerned with the

uptake and transport of nutrients or metabolites and carbo-

hydrate metabolism. Erasmus and Ohman (1963) were of the

view that the alkaline phosphatase was secreted by the para-

s i t e because there was intense reaction in the lamina pro -

pr ia ( just at the s i te of attachment with the host t i ssue)

in many str ig ied trematodes. They discussed the role o f

t h i s phenomenon in relat ion to host-parasite relat ionship.

Contrary to this , another interesting report in the case of

adults and encysted metacercaria of Posthodiplostomum

minimum by Bogitsh (l966a) suggests that alkaline phospha-

tase was produced by the host ' s intest inal epithelium,

Threadgold (l968) i s of the opinion that phosphatases play

13

an important role in dephosphorylation of nutrients a f t e r

they enter the organism from the environment. They may

also dephosphorylate either before or a f ter the nutrients

leave the c e l l and are transported to other adjoining c e l l s

within the organism. According to Erasmus (l970) acid

phosphatase in D. phoxini i s present in the walls of the

adhesive organ chamber, the mic rov i l l i and the c i s temae of

endoplasmic reticulum of the gland c e l l s . He suggested

that th is enzyme might bring about dissolution of the host

t issue at the host-parasite interphase, A review of the

digest ive tract of digenetic trematodes (Bogitsh,1975) sho-

wed that in the organisms studied to date, the gastrodermis

i s e i ther a syncytium or characterized by de f in i te c e l l

boundaries and that acid phosphatase has not only been an

ubiquitous enzyme in gastrodermis but also analogous to

alkaline phosphatase, observed in the vertebrate digestive

system.

Similarly general esterases have also been reported

from the nervous system, tegument, sub tegumental c e l l s ,

excretory system, caecal l ining, suckers, basement layer

o f the testes and parenchyma of a number of trematode spe-

c i e s (Bogitsh,1966b; 6hman,l966; James and Bowers,1967;

Halton,1967a b; Kravica ^ 1967a b ; l 9 7 l ; Barry and

Mawdesiey-Thomas,1968; Halton and Morris,1969; Reznik,1971;

Allen and Harkema,1972; Sood and Gupta,1974; Pat i l and

Rodgi,l976a5Mandawat and Sharma,1978; Roy,1980; Choubisa

et a l . , l 9 8 2 ; Soboleva and Zdarslca, 1983 and Alam and Nizami,

14

1984). The functional role of th i s enzyme group has been

suggested to be hydrolysis of the host tissue during f e e d -

ing (Chappell,1980), while many others agree that the enz-

yme group also help in the absorption and transport of

nutr i t ive and excretory materials across the c e l l membranes

and also in the carbohydrate metabolism. Ohman (l965)

histochemically l o ca l i zed esterases in some s tr ige id t r e -

matodes and found that d i f ferent kinds of enzymes were

secreted to the outside from the lappets and gland c e l l s

of the adhesive organ. In his opinion the l i po id layer

might have been attacked by esterases which help the para-

s i te to dissolve the host tissue at the s i te of attachment.

Other enzymes have rarely been studied histochemi-

c a l l y in detai l and therefore, there occur stray reports

on them, Lee (l952) -showed the presence of leucine amino-

peptidase in the intest inal epithelium o f F. hepatica

while i t was absent in D. dendriticum. The holdfast organ

of Alaria marcianae was also reported to have leucine

aminopeptidase ac t iv i ty (Bhatti and Johnson,1972). ^ - g l u -

curonidase has also been reported from d i f f erent species

o f trematodes (Fripp,l966; Bhatti and Johnson,1972). Cyto-

chrome and monoamine oxidases have been reported to be pre-

sent in d i f ferent regions of P. adenocephala and P. cervi

(Allen,1972; Sharma and Hora,1979). The study on the tegu-

ment of S. mansoni and Haematdloechus medioplexus by Bogitsh

and Krupa ( l97 l ) revealed the presence of nucleoside phos-

phatase act iv i ty in the tegument of both the species . But

15

the other tvro species v i z . , Gorgoderina attenuata and

Megalodiscus temperatus have been reported to have l i t t l e

or no ab i l i ty f o r tegumental transport because o f the absence

o f th is enzyme in their tegument. Wide spread occurrence o f

adenosine triphosphatase (a mitochondrial hydrolytic enzyme)

a c t i v i t y in the t issues o f several species has been shown by

Wheater and Wilson (1976); Sharma (l976) and Michael and

Awadalla { l978) . Similarly s'-nucleotidase ac t iv i ty has

been found in d i f f erent organs part icular ly gonads in a

number of species (Sharma,1976; Michael and Awadalla,1978).

The distribution of glucose-6-phosphatase in the organiza-

t ion of F. gigantica has been reported as uniform through-

out the body (Michael and Awadalla,1978).

Some histochemical studies have also been carried out

on the loca l izat ion of dehydrogenases in trematodes by seve-

ra l workers. In F. hepatica the succinic dehydrogenase seemed

to be insensitive to hexachlorophene and hexachloroethane

(5 ppm and 100 ppm respect ively) and was uniformally d i s t r i -

buted in d i f ferent organs. Further, Allen (l972) reported

the same pattern of distr ibution of succinic dehydrogenase

in P. adenocephala.

The distribution of succinic dehydrogenase in the lym-

phatic system of Ceylonocotyle scoliocoellum has been repor-

ted by Sharma ( l978) . In his opinion the pattern of granules

representing the distr ibution of the enzyme a c t i v i t y , more

or l e s s coincides with the distr ibution of mitochondria,

indicat ing that succinic dehydrogenase i s l o ca l i zed in or

16

near the mitochondria and plays some role in the transport

of certain metabolites.

Succinic dehydrogenase was also l oca l i zed in the c e r -

caria of Cloacitrema michiganensis by Leflore et a l , ( l980) .

The authors opined that some electron transport system

might have been available to the f ree l iv ing stages of the

parasite . Sharma et (l980) detected succinic dehydro-

genase ac t iv i ty in the t issues of G. crumenifer. The more

pronounced reaction was observed mainly in the muscles,

lymph vessels , nerves and v i t e l l a r i a . A l i t t l e a c t iv i ty

was also seen in the gut, excretory canal, uterus, tes tes ,

Mehli 's gland and parenchyma. Gorchilova (l980) also l o c a -

l i z e d the succinic dehydrogenase ac t iv i ty in the intest ina l

walls of mature F. hepatica. He reported that the enzyme

was present usually in the inside layer of the mitochon-

dr ia l membrane in the central zone of intest inal wall .

Allen (1972) reported the presense of malic dehydro-

genase in most of the t issues of P. adenocephala. A high

degree of succinic , malic, isocitr ic ,o<-glycerophosphate

and glucose-6-phosphate dehydrogenase ac t iv i ty has been

reported by Leflore ( l978) from the t a i l , tegument, caudal

pocket, cerebral ganglia and i t s commissures, excretory

bladder, acetabulum and oral sucker, part icularly in the

muscles around the s t y l e t . Only moderate ac t iv i ty was

obtained f o r l a c t i c and 6-phosphogluconate dehydrogenases

at these s i t es . Glutamic dehydrogenase was l o ca l i zed only

in the t a i l and testes while f o r alcohol dehydrogenase

17

these were completely negative. He suggested that several

energy producing sequences may be available to the cercariae .

Malic dehydrogenase was also observed in the d i f f erent regions

o f the cercaria of C. michiganensis (Leflore ^ a l . , l 9 8 0 ) , and

the authors suggested that the t r i carboxyl i c acid cycle (TCA)

might be operative in the cercaria . Malate dehydrogenase was

also ' detected in the reproductive organs and in the muscles,

indicating the high energy requirements in these two ' s i t e s .

Lactate dehydrogenase was studied in the t issues o f

F, hepatica by Thorpe ( l967) . He found that hexachloroethane

(5 ppm and 100 ppm respect ively) did not markedly a l ter the

chemical reaction at the site of enzyme l o ca l i za t i on . Pat i l

and Rodgi (1975b) found that in the t issues of P, c e rv l . l a c t -

ate dehydrogenase was present in the oral sucker, sub c u t i c l e ,

genital cup, parenchyma surrounding the caecum and periphery

o f the testes ^ d ovary. More pronounced ac t iv i ty was seen in

the parenchyma surrounding the caeciom. Only the v i t e l l i n e .

c e l l s were fo\jnd negative f o r the presence o f lactate dehydro-

genase. The authors suggested that Embden-Meyerhof pathway

may be operative in the above case. The cercaria of C. michi-

ganensis was also found to possess the lactate dehydrogenase

a c t i v i t y (Leflore ^ ^ . , 1 9 8 0 ) .

A quantitative study by Dodin et a l . (l956) on the

presence of glucose-6-phosphate dehydrogenase in the v a r i -

ous t issues of mansoni organization revealed that the enzyme

18

a c t i v i t y in the males was 5 times more than that of the f e -

males. They also observed that in the males the main s i t e s

o f ac t iv i ty were sub cutaneous t issue and the skin papi l lae ,

while in females i t was principal ly present in the v i t e l ^ ,

l ine glands. Most of the t issues of R adenocephala have

been reported to have glucose-6-phosphate dehydrogenase

a c t i v i t y (Allen,1972). The l oca l i zat ion and distr ibut ion

o f th i s enzyme was also observed in a l l the t issues except

the v i t e l l i n e c e l l s of P, cervi but the over a l l d i s t r ibu -

t ion was not uniform (Pati l and Rodgi,1976b), The authors

suggested the presence of Embden-Meyerhof pathway in the

metabolic process of the parasite. Leflore a l . (l980) also

found this enzyme in the t issues o f the cercaria of

C. michiganensis and suggested that TCA cycle might be oper-

ative in th is larval form.

The studies on other dehydrogenases have been f r a g -

mentary. Pati l and Rodgi (l976b) reported thate<-glycero-

phosphate and 6-phosphogluconate dehydrogenases were pre -

sent in the t issues of P. cervi mainly in the oral sucker,

sub c u t i c l e , genital cup, parenchyma surrounding the cae-

cum and periphery of the testes and ovary. The strongest

reaction f o r th is enzyme was observed in the parenchyma

surrounding the caec^im while v i t e l l i n e c e l l s did not show

pos i t i ve reaction. Embden-Meyerhof pathway as well as

pentose phosphate shunt was thought to be operative in the

above case. Strong reaction f o r the presence of 6-phospho-

gluconate,«<-glycerophosphate and i soc i t ra te dehyrogenases

19

was observed in the d i f ferent t issues of the cercaria of

C. michiganensis by Leflore ^ a l , ( l980) .

The observation on the occurrence of various enzymes

of g lyco lys is , pentose phosphate shunt and TCA cycle in

the trematodes by histochemical methods and their d i f f e r e n -

t i a l distribution led to suggest that possibly the three

cyc les are present but the functional role in the trematode

metabolism i s s t i l l debatable.

Isoenzyme studies;

Some important enzymes of carbohydrate metabolism,

mainly belonging to the TCA cycle , pentose phosphate shunt,

and g lyco lyt i c pathway have been studied biochemically in

a number of trematode species by various workers (Prichard

and Schofield, 1968; DeZoeten et ,1969; Senutaite, 1969,

1973; Strum et ^ . , 1969 ,1972 ; Vaatstra,1969; Kravica ^ al.,

1971; Shishov,1971; Srivastava et ,1971; K6hler,l972;

Coles,1973; Kohler and Hanselman,1973; Saxon and Dunagan,

1975; Shestak,l975; Vykhrestyuk and Klochkova,1977;

Vykherstyuk et ^ . , 1 9 7 7 ; Smith and Brown,1977; Yusufi and

Siddiqi,1978; Van Vugt,1980; Umezurike and Anya,1980; and

many others) . Most o f the authors have suggested that the

embden-Meyerhof pathway, pentose phosphate shunt and TCA

cyc le might have been operative in some form or the other

in the metabolism of the worms.

Conde-Del Pino et a l . ( 1 9 6 6 ) compared the malic and

l a c t i c dehydrogenase isoenzymes of cercariae and adult of

20

S, mansoni. Two of the isoenzymes of malic dehydrogenase

were fotind d i f ferent in the cercariae and adults of the

worm. Further, considerable d i f ferences were noted in the

mob i l i t i e s of the isoenzymes of the host and the parasite

t i s s u e . Only one band of l a c t i c dehydrogenase was found

in the males and females of S. mansoni but none in cerca -

r iae .

Electrophoretic separation and comparision of i soen-

zyme p r o f i l e s of acid phosphatase, non-spec i f i c esterase,

leucine aminopeptidase and lac tate , malate, ^^-glutamate,

glucose-5-phosphate and 6-phosphogluconate dehydrogen-

ases of 4 populations o f S. mansoni and 1 population of

S, haematobium by Coles ( l97la) revealed notable d i f f e r e n -

ces between the two sexes of S, mansoni f o r esterase, l a c -

ta te , malate and glucose-6-phosphate dehydrogenases. In

the case of S, haematobium the d i f ference was found betw-

een esterase and lactate dehydrogenase of the males and

females while between the two species, esterase, lactate

and glucose-6-phosphate dehydrogenase isoenzymes showed

considerable d i f ferences , Oya et (l970) found that

S, mansoni possesses 4 isoenzymes of MDH while S, .laponicum

has only three. No di f ference was noticed in the isoen-

zyme pattern of the d i f ferent sexes of the two species .

The east African strain of S. mansoni has 2 to 4 i soen-

zymes of malate dehydrogenase as reported by Coles (1971a).

He reported variations in the two fast moving bands

21

within a small community, sometimes variations were noted

even between worms in one host. But in another study

XColes, 197113), no s igni f i cant d i f ferences in the isoenzymes

of malate, lactate and glucose-6-phosphate dehydrogenases,

non-spec i f i c esterases and acid phosphatase of 8 d i f f erent

strains of S, mansoni have been found. Electrophoretic

observations on the MDH isoenzymes o f the d i f ferent stages

of S. mansoni by Homewood et ( l973) revealed 2 i d e n t i -

ca l bands in males as well as in females and even in the

mixed extracts of both the sexes.

I soe lec t r i c focussing f o r the determination of l a c t -

ate and malate dehydrogenase isoenzymes in Schistosoma spp,

by Ross (l976) led him to suggest that such studies may be

potent ia l ly more useful f o r the determination of s p e c i f i c

and intraspec i f i c d i f ferences in schistosome sp. complex.

Yan et, eQ , (1976) studied the malic dehydrogenase isoenzy-

mes of the d i f ferent strains of S. .japonicum and sub-species

o f Oncomelania hupensis, (O.h* quadrasi and O.h, l lndoensis)

and reported considerably d i f ferent isoenzyme patterns. The

isoenzymes of lactate and malate dehydrogenases and acid

phosphatases in Schistosoma spp. were studied by. Ross (l976).

S igni f i cant i n t e r - and in t raspec i f i c d i f ferences have

been noted part icularly in the IJ)H isoenzyme pattern while

MDH and acid phosphatases were suggested to be useful over

a l imited range of the group. Le Riche and Sewell (l977)

distinguished d i f ferences between Taenia saginata and

22

T, solium on the basis of glucose phosphate isomerase i s o -

enzyme patterns of the two species . Isoenzyme studies of

MDH, LDH and acid phosphatase have also been carried out

on d i f f e rent schistosome spp. by Ross £t ( l978) . The

authors suggested that the malate dehydrogenase isoenzyme^^

system was not sat is factory and that lactate dehydrogenase

isoenzymes were more useful at the in terspec i f i c than at

the intraspec i f i c l e v e l . Further they were of the view

that i f more enzyme systems are included in a study, the

more balanced wi l l be the overal l assessment of i n t e r r e l a -

t ionships . Differences between cytoplasmic and mitochondr-

i a l enzymes have been reported by Rotmans(i978) a f t e r stu-

dying the malate dehydrogenase isoenzymes of S, mansoni

through i s o e l e c t r i c focussing. Similarly Wright et

(1979) studied 7 enzyme systems in adult worms from 2 i s o -

l a t e s of ^ intercalatum (one from Zaire and the other

from the Cameroons). The d i f ferences were noted in the

lac tate dehydrogenase isoenzymes which are more profound

in the case of glucose-6-phosphate dehydrogenase and phos-

phoglucomutase (the last system d i f f e red in the adults as

well as in the cercar ia l extracts ) . Umezurike and Anya

(1980) reported 5 isoenzyme of lactate dehydrogenase in

F. hepatica. They further noticed the predominance of LDH-1

2 and 3 while LDH-4 and 5 were in traces . At least 5 multi-

ple forms of phenol oxidase in F. hepatica have been repor-

ted by Nellaiappan and Ramalingam ( l980) .

23

Agatsuma and Suzuki (.1980) studied the isoenzymes

o f some enzymes of common l i v e r f luke Fasciola sp . . They

observed two forms of adenylate kinase and two forms of

phosphaoglucomutase which were almost similar in the i r

e lectrophoret ic mobil i ty , but phosphogluconate dehydroge-

nase and esterase showed no var iat ions . Glucose phosphate

isomerase, NAD dependent glutamate dehydrogenase and NADP

dependent malic enzyme of Fasciola sp. from Japan have

been studied on starch gel by Agatsuma ( l 98 la ) . He did

not f ind variations in the banding pattern f o r any of the

enzymes from individual f lukes .

In a similar study Agatsuma ( l98 lb ) showed 4 d i f -

ferent phenotsrpes in the natural populations of adult ,

P, i loktsuenensis on the basis of isoenzyme pattern of

glucose phosphate isomerase. Agatsuma ( l98 l c ) compared

the a l l e l i c frequencies of glucose phosphate isomerase iso-

enzymes in natural populations of Paragonimus sp, v i z .

P, oh ira i and P, sadoensis, obtained from d i f ferent parts

o f the country. He recorded phenotypic s imi lar i t i es and

d i f f e rences on the basis of isoenzyme patterns.

To distinguish the two types of Anisakis larvae,

glucose phosphate isomerase and phosphoglucomutase i soen-

zymes were observed by Agatsuma ( l 9 8 l d ) . No variat ions

were found between the two and therefore , he suggested

that i t might be due to the occurrence of random mating

in groups of each type, Agatsuma and Suzuki ( l 98 l )

24

reported that the isoenzymes of adenylate kinase, esterase,

phosphogluconate dehydrogenase, hexokinase, glucose phosph;--

ate isomerase and glucose-6-phosphate dehydrogenase of

P. ohirait P, miyazakii and the rat (host) t i ssue , d i f f e red

in their e lectrophoret ic mobi l i t ies and at least 4 enzymes

(adenylate kinase, phosphogluconate dehydrogenase, glucose

phosphate isomerase and glucose-6-phosphate dehydrogenase)

have been found to d i f f e r in number and'mobil it ies of bands,

even between the two parasite species .

Biochemical characterization of Trypanosoma sp. has

successful ly been carried out by Richard and Octavio ( l98 l )

through enzyme electrophoresis . In a l l , 13 enzymes were

compared f o r cultures of T. avium, T. vesper t i l i on i s ,

T. cruzi and T, rangel i . The d i f f erences and s imi lar i t i es

(isoenzyme types) in the nutnber and pattern of the i soen-

zyme bands f o r each enzyme between d i f f erent species were

described to be due to their geographical d is tr ibut ion .

Fi fteen cercarial enzymes of S. mansoni and S. rodhaini

were studied by Fletcher et ( l 9 8 l ) . Eight enzymes have

been found to have similar isoenzymes while another seven

enzyme showed considerable d i f ferences in the nTomber and

mobi l i t i es of bands. According to th is study the best

diagnostic d i f f e rent ia t i on was obtained with phosphogluco-

mutase which existed in 3 a l l e l i c forms. Fletcher and

LoVerde ( l98i ) analysed 14 enzyme systems of S. mansoni

as markers associated with genes f o r i n f e c t i v i t y to snai ls .

25

They have mentioned a correlation between the strains of

low and high i n f e c t i v i t y f o r lactate dehydrogenase pheno-

type frequencies. Glucose phosphate isomerase and,lactate

dehydrogenase isoenzymes, in d i f ferent species of f i l a r i a l

worms have been studied by Lim et a l , ( l 9 8 l ) . They obser -

ved di f ferences and suggested that c l o se ly related species

might easi ly be distinguished from each other with the help

o f isoenzyme studies.

Southgate et a l , ( l 98 l ) studied the isoenzyme pattern

of phosphoglucomutase, glucose-6-phosphate dehydrogenase,

acid phosphatase, glucose phosphate isomerase, malate and

lac tate dehydrogenases in d i f ferent schistosome species .

Minor to d ist inct d i f ferences in the isoenzyme patterns of

d i f f e r e n t enzymes were noted at i n t e r - and in t raspec i f i c

l e v e l . The isoenzyme patterns of glucose phosphate isomer-

ase and phosphoglucomutase of hydatid cysts (Echinococcus

granulosus) protoscoleces from human, camel and sheep have

been analysed by Macpherson and McManus ( l982) . The i soen-

zymes have been found consistently with negl ig ib le a l t e r -

at ions . Hermoso et a l . (l982) compared the isoenzymes of

glucose phosphate isomerase and lac tate , malate and glucose

6-phosphate dehydrogenases of Moneizia eypansa and Avite l l ina

centripunctata. They fovind d is t inct d i f ferences in the

number of. isoenzymes in the gravid as well as sexually mat-

ure proglott ids from both the worms, which were further

confirmed on subsequent i s o e l e c t r i c focussing of the same

26

material , de Boissezon and Jelnes (l982) showed d i s t i n g u i -

shable di f ferences in the isoenzymes of phosphoglucose i s o -

merase, phosphoglucomutase, hexokinase, adenylate kinase,

fructokinase, mannose-6-phosphate isomerase, glucose-5-phos-

phate dehydrogenase and malate dehydrogenase in d i f f e r e n t

i s o l a t e s of cercariae and adults; individual; and sexes o f

S. mansoni and S, rodhaini. This led them to suggest that

such charactersties might be useful in quick ident i f i ca t i on

o f schistosome cercariae emerging from Biomphalaria sp. in

A f r i ca . Flockhart (l982) ident i f i ed some Onchocerca sp.

namely 0, gutturosa. 0 . gibsoni and 0 . l i e n a l i s on the basis

of isoenzyme analysis.

Agatsuma and Suzuki (l983) studied isoenzyme patterns

o f glucose-6-phosphate dehydrogenase, glucose phosphate i s o -

merase, hexokinase, malate and 6-phosphogluconate dehydro-

genases and phosphoglucomutase in the adult males o f S.

.japonicum and S. mansoni. The number and posit ion of bands

was found d i f ferent between the two species as well as of

the host (mouse) blood and muscle. I t was then concluded

that S. mansoni was c lear ly distinguishable from S. .japoni-

cum at the molecular l e v e l . Further, isoenzyme pattern of

lac tate and malate dehydrogenases of G, crumenifer from

two d i f ferent hosts (buffalo and sheep) have been studied

by Dhandayuthapani et ( l983) , who suggested that the

enzyme configuration of the parasite might be influenced

d i f f e r e n t l y by d i f f erent hosts.

27

The whole worms homogenates have also been studied by-

several workers to obtain the d i f f e r e n t i a l protein pattern

o f the di f ferent species through electrophoresis and i s o e l e c -

t r i c focussing. Since the protein composition of an orga-

nism r e f l e c t s i t s genetic const i tut ion, separation of p ro -

t e i n s may prove to be a valuable taxonomic too l in studies

o f a variety of parasites . Yoshiraura et ( l970) o b t a i -

ned 21 bands, 19 o f them reproducible from the whole worm

homogenate of P. k e l l i c o t t i . The densitometeric observation

revealed 8 characterstic peaks. The protein pattern of

P. k e l l i c o t t i was found to be d i f f e rent from P. westermani.

P. oh ira i . P. i loktsuenensis and P. miyazakiL P. k e l l i c o t t i

and P. mivazakii the two morphologically similar species show-

ed similar patterns near both ends of the gel but the mid-gel

area showed d is t inct d i f f erences . Klimenko and Velichko

( l972) studied the protein spectrum of d i f ferent amphistomes.

Dif ferences have been noticed in the protein spectrum of

Calicophoron calicophorum and Liorchis scotiae from G. cru-

men i f er« Spec i f i c and d is t inct protein p r o f i l e s were demons-

trated by i s o e l e c t r i c focussing of soluble whole body p r o t -

e ins of four Di-phvllobothritim species, in which altogether

3 1 - 3 6 protein bands per species were obtained by Byliind

and Djupsund (l977),The authors suggested the use o f sens i -

t i v e chemotaxonomic methods to help characterize and delimit

species in the troublesome Diphyllobothrium group.

The va l id i ty o f the three species o f trematodes v i z .

P. microbothrium, P. cerv j and P. microbothriodes was

28

confirmed by Osikovoski et ( l978) , on the basis o f the

patterns of water soluble protein extracts of the three spe-

c i e s . Choi e^ a l . ( l 98 l ) compared the protein composition

o f various parts (body wall, genital organs, digest ive organs

and body f l u i d ) of A. suum, on the basis of the number o f

bands obtained f o r each of them. Forty three protein bands

in the body wall, 51 in genital organs, 47 in digest ive orga-

ns and 34 in body f l u i d have been recorded. Some bands of

s p e c i f i c molecular weight were shown as unique to the i r r e s -

pect ive organ. The host-parasite protein spectrum was s tu -

died by Sekretaryuk (l982) in the case of Philometroides

in fec t i on in carps. The protein fractions of in fected carp

and that of P. lusiana were fotmd similar.

Atkinson and Atkinson (l982) reported that one and two

dimensional separation of S, mansoni proteins (synthesized

in v i t r o ) by PAGE and flurography showed s imi lar i t i es and

that homogametic males produced 5 polypeptides while hetero-

gametic females did not show the same pattern. The protein

pattern of gravid as well as mature proglott ids of M. exTPansa

and centripunctata have been found to d i f f e r considerably

(Hermoso ^ a l . . 1982).

I t i s evident from the foregoing review of the l i t e r a -

ture pertaining to this study that there s t i l l ex ist many gaps

and lacunae in our knowledge of trematode biochemistry and

physiology and that some fascinating aspects l ike host-para-

s i t e relationship, biochemical taxonomy and general metaboli-

sm are s t i l l in need o f further extensive invest igat ion.

M A T E R I A L S A N D M E T H O D S

29

Col lect ion of parasites :

The amphlstome parasites, G. crumenlfer and P. eplc l l - -

tum from the rumen and G. explanatum from the l i v e r of

Indian water bu f fa lo , Bubalus bubalis were co l l e c ted from

the l o c a l slaughter house. The parasites were brought to

the laboratory expeditiously in a thermos f lask along with

in fec ted host t i ssue .

The trematodes were detached from the host t issue and

washed with Tyrode's sal ine. Pieces of rumen and l i v e r were

also washed in the same manner.

Routine paraf f in technique was used to l o c a l i z e the

fo l lowing various biochemical components in d i f f erent r e g i -

ons of the parasites . Cryotomy was carried out f o r the loca-

l i z a t i o n of various enzymes.

The various biochemical components were acid mucopoly-

saccharides, amyloids, collagen, e las t in , f i b r i n , nucleic

acids (DNA and RNA) and tryptophan (Table I ) .

Paraf f in technique;

Live worms were immediately washed twice with the

Tyrode's solution. For f ixat ion , the worms were put betwe-

en two sl ides londer sl ight pressure and kept in a petr i -d ish

containing the desired f i x a t i v e . The f i xa t ives used f o r

general histochemistry were acet ic formalin alcohol (AFA),

30

Carnoy's f l u i d and 10% buffered formalin. The specimens

were kept in d i f f e rent f i xat ives from 4 h to overnight. The

most suitable f i x a t i o n was found as f o l l ows : 10 h in AFA,

4-6 h in Camoy's and overnight in 10% buffered formalin.

After f ixat ion the parasites were properly dehydrated in the

ascending grades of alcohol (30%-100%), The specimens were

passed through 96% and absolute alcohol twice f o r the same

duration. After dehydration they were cleared in two chan-

ges of xylene f o r 10 minutes each and then transferred to a

mixture of 1:1 paraf f in and xylene f o r i n f i l t r a t i o n and kept

at 56* 0 f o r 2 h. Embedding was carried out in pure molten

para f f in . Blocks of each parasite were made .and trimmed.

The sections, 5-7 yu thick were cut on a rotary microtome

(E.Leitz , Wetzlar).

The ribbons of ser ia l sections were l i f t e d on prealbu-

minized s l ides . The ribbons were streched by putting a few

drops of water below them and keeping the s l ides on a modera-

t e l y warm hot-plate . The excess water was drained o f f and

the s l ides were allowed to dry at room temperature. The sec-

t ions were then stained in d i f ferent staining mixtures, f o r

the loca l i zat ion of d i f ferent biochemical components. The

de ta i l s of staining solutions are given in Table I .

Cryotomy;

Fresh material was used f o r the loca l i zat ion of enzymes

in the frozen sections of the parasites . Tissue-Tek I I com-

pound (Lab-Tek Products, U.S.A.) was used f o r embedding with-

out pre f ixat ion . Frozen sections, 10-12 f j thick were cut on

31

a cryostat (AOC, Mod.NO. 840, N.Y. ,U .S .A . ) . During s e c t i o n -

ing the temperature of the inner chamber was kept at -20°C,

The sect ions were l i f t e d on clean glass s l ides d i r e c t l y from

the k n i f e . They were allowed to dry by keeping them in the

cryostat chamber f o r a while. The sect ions thus obtained

were immediately treated in d i f f e r e n t appropriate so lut ions

as mentioned below in Appendix A.

Control ;

In histoenzymological l o c a l i z a t i o n and in the isoenzyme

p r o f i l e of every enzyme a contro l was nin simultaenously in

order to compare the resu l ts . For contro l , appropriate meta-

b o l i c inhib i tors (competetive and non-competetive) were used

as well as heat treatment was given or appropriate substrate

was omitted from the staining so lut ion . The various i n h i b i -

t o r s used are given in Table I I .

Microphotography;

Microphotographs of the d i f f e r e n t i a l l o c a l i z a t i o n o f

d i f f e r e n t enzymes in the parasites were taken with the help

o f a t r inocular microscope (Getner, India) . Asahi-Pentax

(K 1000) camera and 125 ASA black and white f i lm was used.

In order to avoid the expensive cost o f photographs,

some se lected ones, showing important regions of l o c a l i z a -

t i o n o f d i f f e rent enzymes are appended in the present thes i s

and the deta i l s of the results are given in the form of

t a b l e s at proper p laces .

32

Chemicals;

The chemicals used f o r PAGE and histochemistry and

histoenzymology were obtained from Sigma Chemical Co. ,U.S.A.,

Kochlight Laboratories, England; Edward Gurr, England; BDH

(AR) Chemical Co., England and Loba Chemicals, Bombay, India.

Preparation of homogenate:

Glass te f lon homogenizer was used to prepare 25% and

20% (w/v) homogenate of each parasite and the host t issue

respect ively at 4°C. The homogenates were kept in the r e f r i -

gerator f o r 2 h before being centrifuged. The larger p a r t i -

c l e s of the tissue debris thus set t led were discarded and

the supernatant was then centrifuged at 30,000- 40,000 rpm

in a refr igerated ul tra centrifuge (Jaetzki, VAC 60, GDR)

f o r 30 min at 4°C. The homogenates were kept in the deep

f reeze at -10®C, The quantitative determination of protein

was carried out by the method of Bradford ( l976) .

Polyacrylamide gel electrophoresis (PAGE):

Polyacrylamide gel e lectrophoresis was carried out by

using the standard method of Davis (l964) f o r obtaining the

separations of the isoenzyme p r o f i l e .

The gels were cast in corning glass tubes measuring

75 mm in length and 5 mm inner diameter. The tubes were

thoroughly cleaned with chromic acid and detergent. They

were then allowed to dry in the thermostat at 80°C.

33

The glass tubes were f i t t e d in the gel casting stand,

v e r t i c a l l y erect with the help of holders. The lower ends

of the tubes were sealed by using rubber bungs to stop any

leakage. The polyacrylamide gel solution ( f o r de ta i l s see

below) was then poured into the tubes with the help of a

hypodermic syringe. While pouring the solution, care was

taken to avoid trapping of a i r bubbles in the gel so lut ion.

The gels were polymerized f o r about 30-40 min under d irect

f lourescent l i ght .

Electrophoresis was carried out in 65 mm high small

pore gel of 1% acrylamide concentration at a pH of 8 .8 -9 .0

at 4°C. The large pore gel was found unsuitable,possibly

because of the high l i p i d content in the homogenates, there-

f o r e , the spacer gel was not used in th is study e i ther .

The homogenate samples containing 60-80 ^g protein,

mixed with a drop of bromophenol blue (tracking dye) was

applied on top of the gel . The sample layer was covered

with chamber buf fer and electrophoresis was conducted using

a power supply (Toshniwal CMOI 3 ) . A current of 3 mA per

gel was applied unt i l the tracking dye, bromophenol blue

(BPB) had moved 60 mm towards anode. At the end of the run

the tubes were taken out from the assembly and the gels were

removed from their respective tubes with the help of a syr -

inge and a 24 gauge hypodermic needle. The d i s t i l l e d water

was pushed with pressure into the interphase between the gel

and the inner margin of the glass tube. In this manner the .

34

ge l s were taken out smoothly. Immediately a black nylon

b r i s t l e was inserted in the middle of the BPB band in each

g e l . This served as a marker i f the tracking dye d i s appear-

ed from the gel during incubation in d i f f e rent incubation

media.

Composition o f acrvlamide gel so lut ion ;

Solution-A

IN HCl Tris

/ / TEMED (l^NN-Tetraethyle

et'Hylene diamine) D i s t i l l e d crater to

48.00 ml 35.69 g

0.23 ml 100,00 ml

Solution-B

IN HCl Tr is TEMED D i s t i l l e d water to

48.00 ml 5.98 g o,46 ml

100.00 ml

Solution-C

Acrylamide

Bis (N,/Methylene b i s acrylamide)

D i s t i l l e d water to

28.00 g ( f o r 7% c onc . ) or 40,00 g ( f o r 10% c o n c . )

0.735 g lOOoOO ml

Solution-D

35

Acrylamide

Bis D is t i l l ed water to

10.00 g ( f o r 2.5% conc . ) or 14.00 g ( f o r 3.3% conc . ) 2.5 g

100.00 ml

Solution-E

Riboflavin Dis t i l l ed water to

4.00 g 100.00 ml

Solution-F

Sucrose D i s t i l l ed water to

40.00 g 100.00 ml

Small pore gel so lut ion :

1 part A 2 parts C 1 part

This solution was mixed with an equal quantity of

0.14% ammonium persulphate solution.

D i s t i l l e d water (pH 8 .8 -9 .0 )

Large pore gel solut ion:

1 part 2 part5 1 part 4 partfi

B D E F (pH 6 .6 -6 .8 )

36

Stock solution f o r buf fer chambers:

Tris 60OO g Glycine 28.80 g Dis t i l l ed water to 1000.00 ml (pH 8 ,3)

One tenth concentration of the above buf fer solution

was used in the chambers of the electrophoretic assembly as

e l e c t r o l y t e .

SDS polyacrylamlde gel electrophoresis f o r general proteins:

In order to compare the proteins of the three species

o f amphistomes and infected host t issue the sodium dodecyl

sulphate gel elestrophoresis (SDS-PAGE) was carried out in

glass tubes (1O.5 cm long with 0,5 cm internal diameter).

Seven percent acrylamide gels were prepared from the stock

solution of 30% acrylamide and 0,8% Bis. The f i n a l concen-

trat ion of the gels contained 0,375M tris-HCl (pH 8,8) and

0,1% SDS. The gels were polymerized through chemical action

by adding 0,025% (by volume) TEMED and ammonium persulphate.

The chamber buf fer ( t r i s -g lyc ine pH 8.3) containing 0.025M

t r i s , 0,192M glycine and Oo1% SDS was used as e l e c t r o l y t e .

Homogenate sample of each parasite and the host t issue was

heated to 100°C f o r 3-5 min a f ter mixing with a solution

which contained 0.062M tris-HCl (pH 6 , 8 ) , 3% SDS, 10% suc-

rose and -mercaptoethanol. The marker dye BPB (a drop

of 0.01%, solution) was added to each sample and a 100 yul

sample containing 60-80 jsg protein was applied. The electro-

phoresis was conducted at a constant current (3 mA per gel)

37

unt i l the BPB band migrated up to the desired l e v e l , towards

anode. The complete electrophoretic run took about 2 h. The

gels were removed from the electrophoresis assembly. Before

staining, the gels were f ixed in perchlor ic acid (PCA), metha-

nol and d i s t i l l e d water (5:20:75 v / v ) f o r 30 min.

After f ixat ion the gels were kept in the Coomassie

b r i l l i a n t blue (CBB) G-250 staining solution (40 mg dye d i s -

solved in the f i x e r solution/100 ml) f o r 2 h. Stained gels

were transferred to the destaining solution (g lac ia l acet ic

acid 5 parts, methanol 20 parts and water 75 parts ) . After

desired destaining the protein bands appeared. The gels were

stored in 7% acet i c acid solution. Densitometery was then

performed as f o l l ows :

Staining:

The d i f ferent staining methods used f o r d i f f e rent enzy-

mes were as described by Shaw and Prasad ( l970) , Pearse (l968)

(see Appendix A) and f o r general proteins the method of

Laemmli (l970) was used. The staining of gels was carried out

at 37°C in a thermostat.

Photography of ge l s :

The gels showing isoenzyme and protein patterns as e v i -

dent by d i f f e r i n g relat ive mobi l i t ies with s p e c i f i c staining

were photographed by Olympus-OM iO (Japan) camera, using 80 ASA

black and white f i lm. The gels were put between the camera

and a di f fused f lourescent l ight source. For each snap the

cathodal end was kept at the top while anodal end was always

38

at the bottom.

Densitometry and quantitation o f isoenzyme p r o f i l e ;

The densitometry f o r quantitation of the coloured

bands a f t e r appropriate staining was carried out through a

Systronic densitometer. The gels were placed on the adaptor

in a horizontal pos i t ion . The percent transmission was r e -

corded at 1 mm interval throughout the length of the gel up

to the BPB marker. The densitometer was calibrated to 100?

by passing the l ight through the unstained portion below the

marker which served as blank. The red f i l t e r was used agai -

nst the blue bands in the gels .

Nomenclature of isoenzymes;

The isoenzyme pattern observed on the gels was numbered

according to the lUB recommendations (Webb,1964). The enzyme

band nearest to the anode was considered as number 1.

39

Q

m CO

a H O

^

O CO M •X

tt)

0) o c 0) u Q)

«H CD OC

0) tiO J C a* •H -H • p CO o -p cu CO E-1

hO X) -d I W

00 0

cu CO

cd 

cu to ^ CO Q)

o -d

1 >> H o to CO w ft CU J o o -d •H C O -H to S CU 2 ^ -d CU C a CO •H ^ O ^ o O ft •d o H o S •H O •H O O CO

m o <t, c«

o CO _ o cncn c cTv - t- cO «-

o -d

cu C C o •H 'H •H to H •P -P X! •H 0 X) cu •• iH -d <H >> c H fl cu u CO >> O -H XI O o •H XJ tiO to cu H CU o fl ft Ti SM H ^ H 0 O CU H cu CO H S XI

CD VD 0

cu to ^ CO 0) a,

cu •H -P •H pq

CD

CU ID u CO CU CM

I g 0

a H -H ^

^ O H U J) >1

C CU 0

SPU fa

O •d

o TJ

• • to -d •H c c O a CO s C CU bO a o •H T:) § CO -P •H 4J CU H to ft >> H H CO ft >> O C O H •H P § O w EH a «

40

TABLE I I INHIBITORS USED FOR ENZYME CONTROL

S.No. Inhibitors Enzymes

1. Phenyl hydrazine Gluco se-6-pho sphate dehydro genase

2. Sodium arsenate Malate dehydrogenase

3. Malonate Succinate dehydrogenase

4. Aoetaldehyde Alcohol dehydrogenase

5. None Nothing dehydrogenase

6. Sodium arsenate o^-glycerophosphate dehydrogenase

7. Parachloromercuric-benzoate (PCMB)

Glyceraldehyde-3-phosphate dehydrogenase

8. Sodium arsenate Hexose-6-dehydrogenase

9. Sulphanilamide 6-phosphogluconate dehydrogenase

10. Pyrophosphate Glucose phosphate isomerase

11. Sodium f luor ide Phosphoglucomutase

12. Suramin Hexokinase

13. Sodium arsenate Fructose 1,6-diphosphatase

14. <^<-dipyridil Aldolase

R E S U L T S A N D D I S C U S S I O N

41

General Histochemistry:

The d i f f e r e n t i a l distribution of some important b i o -

chemical components l ike acid mucopolysaccharides, amyloids,

col lagen, e last in , f ibrin,tryptophan and nucleic acids (DNA

and RNA) in d i f f e rent regions of the body of the three species

of amphistomes i . e . , G. crumenifer. P. epiclitum and G, expla~

natum of buf fa lo have been presented in Tables I I I - IX .

The acid mucopolysaccharides in the case o f G, crumeni-

f e r are intensely present in the outer layer of the tegument

and in lesser amount in the parenchyma, caecae, in teres t ia l

t i s sue , lymph vessels and in the tegument surrounding the suc-

kers. The tegument and suckers of P. epiclitum possess mode-

rate amounts of acid mucopolysaccharides. The other regions

show almost similar intensity of staining and pattern of d i s -

tr ibution as observed in G. crumenifer. G. explanatum shows

quite a d i f ferent pattern of acid mucopolysaccharide d i s t r i -

bution, In th is case the tegument has l i t t l e acid mucopoly-

saccharides while parenchyma and lymph vessels are negative.

The other s igni f icant di f ference from the former two species

i s the higher concentration of acid mucopolysaccharides in

tes tes and uterus and moderate amounts in the caecae and

eggs (Table I I I ) .

Mucopolysaccharides have e a r l i e r been reported from the

cyst wall of the metacercaria of F. hepatica. Nanophyetus

> s H O -d ft CO o <H o o o (U ^ CO S CO o S CU -p •H •H • O CO CO CO CO • JM > «H CO O ft

• c CO o • •H o •P CO N • •H too H 0) CO H to O . H O ^ H HJ SJ CU

bO tJO w +3 D -P •H >

I 0) o 0 CO 4J 3 CO S H I C CO 0) a CO X! o

n •d CO a 8

> o

>

CU

-p c CU E 0) E-i

03 O

0) •H CO o OJ XJ C •H •H -p +> 0) x: P •H o e p Q) a fn a CO +5 h CO to o Jh CO CO CO CU CU CU

+ + + + +

+ + +

+ +

QJ H O O O

"cO

•H O 0)

c 0 •H CO bO Q) is; 11

CD is: II +

0) -P CO U Q) n o s

+ +

OJ CO a 0) -p

+ + +

43

salmlncola and Parorchis acanthus (Dixon and Mercer,1964;

Dixon,1965; Rees,l967; Porter,1970). The cercaria of

S, manson! has been found to contain mucopolysaccharides

in the acetabular gland (Stirewalt, 1959; Smith £t , 1969).

Further" the tegument of adult F. hepatica« Alarla alata,

Echinostoma revolutum« C, bushinensis. Liorchis scot iae ,

Eurytrema coelomatlcum, D. lanceolatum. 0. fellneus« F,

gigantica, Labostoma ringens and H, ovis have been d e s c r i -

bed to possess intense amount of acid mucopolysaccharides

(Monne,l959; Cheng and Burton,1966; Nacheva,1975,1977;

Molfi ,1976; Halton and Hendrix,1978; Sharma and Mandawat,

1979; Soboleva and Zdarska,1983).

The present f inding on the l o ca l i zat ion and d i s t r i -

bution of acid mucopolysaccharides, part icularly in the

tegument of G. crumenifer and P. epiclitum i s in agreement

with the ear l i e r reports on the tegximent of d i f ferent adult

worms. The presence of this substance in higher concentra-

t ion in the tegument of trematodes inhabiting rumen, i s pro -

bably a biochemical adaptation , to protect the worms from

the hos t ' s immunological reaction. Acid mucopolysaccharides

may also help the parasites in the ir osmoregulation against

continuous changes in the osmotic and chemical environment

in the rumen, caused by promiscuous feeding of the host.

In the case of G. explanatum, considerably low amount o f

acid mucopolysaccharides may be due to the influence of the

t o t a l l y d i f ferent habitat. This parasite inhabits the b i l e

ducts where i t i s continuously exposed to the b i le secretion

44

(containing b i le s a l t s ) , which might be acting as surface

detergent, consequently resulting in the depletion of acid

mucopolysaccharides in the tegument.

Another biochemically important substance are amyloids

which have been moderately present in the outer layer of the

tegument and in the caecae of G. crumenifer. The subtegumen-

tary c e l l s and ventral pouch show very weak reaction. In

P. eplclitum the distribution of amyloid deposition i s more

or l ess the same as in G. crumenifer. The caecae and testes

of G. explanatiM show moderate and weak staining respect ively

while the tegument and other organs are negative (Table IV),

In chemical terms, amyloids are combinations of proteins

with acid polysaccharides and are usually seen as massive

deposits , depending upon the duration of the disease, p a r t i -

cularly in inflammatory diseases. They are further c l a s -

s i f i e d as primary and secondary amyloids on the basis of s t a i -

ning reaction d i f ferences . Amyloids are microscopically homo-

geneous, translucent, acidophil ic substances which usually

occur as extracel lu lar deposits, Pearse ( l % 8 ) described

amyloids as resistant to attack by proteo lyt i c enzymes.

Hence the moderate presence of amyloids in the d i f f erent r e g i -

ons of G. crumenifer and P. epiclitum and almost t o ta l absence

in G, explanatum appears relevant because of the di f ference

in the i r respective haitats . The former two worms inhabit

the rumen and are therefore, lander attack of proteo ly t i c enzy-

mes while the l a t t e r faces the least p o s s i b i l i t y of being a t -

tacked by them in the l i v e r .

CO O H -c) -H •H -p W O W H 3 I I EH CO

tH <H O O G w O 

m bO bO w -p D

-p •H >

I 0) P U o 3 CO +>

I C Cfl OJ S

P O

to •d CO fi 5

> o

cu >

0) CO o (U CO o

CO >

CO

o

bfl 01 EH M • H

rQ H 73 0) CO O

1H U 0) CU •P CO

O H

 H -p H •P CO M •H to 0 0 Xi C •H •H 0 +> CU P 0 H 0 B P CU -P p Sh c X CO CU CQ 0 ^ w CO CO tH C5 C5

B 0 CU CQ +> O

0)

CO bO CU II

CO

CU -p CO J-. CU TZJ i

+ +

CU 03 C CU -p

+ + +

46

Among the structural proteins, collagen has been found

moderately in the subtegumentary c e l l s , oral sucker and testes

of G, crumenifer while the other regions show very weak s t a i -

ning, The l oca l i zat ion of collagen in the d i f ferent regions

of P. epiclitum i s some what similar to G. crumentfer except

that the subtegumentary c e l l s are weakly stained. In the case

of G. explanatum, moderate staining i s observed in the sub-

tegumentary c e l l s , ventral sucker, testes , parenchyma anf lym-

ph vesse ls while the other regions possess very l i t t l e c o l l a -

gen. Apparently there i s no s igni f i cant variation in the d i s -

tr ibut ion of th is structural protein among the three species

(Tabla V).

Though Frandsen (l966) and Anya (l966) described the

presence of collagen in the cut i c l e and egg shel ls of certain

nematodes, in the case of trematodes, material resembling c o l -

lagen has been reported from the basement membrane of F. hepa-

t i c a , E. revolutum and Paramphistomum sp. (Monne,1959). Alth-

ough collagen f i b r e s have not been ident i f i ed with the e l e c -

tron microscope, the collagen l ike material was diastase resis-

tant and PAS pos i t i ve . Bell and Smyth (l958) observed that

the protein of trematode egg may belong to the collagen group

of structural proteins. The collagen of d i f ferent helminths

has been found to have d i f ferent biochemical composition

(von Brand,1973). The collagen iso lated biochemically from

F. hepatica was demonstrated to show di f ferent properties to

that obtained from Ascaris (Nordwig and Hayduk,1969). It has

w

EM O

0) B 0 - p D3

o 1 3 CM

VI

c o Q) -H tiO -H a w H 0 H S O O

O O C O •H

CQ 0) +3 •H

CO CQ N C5J •H H Cd Cd ft O o

CQ t>0 flO w

-P •H

I CD o CQ -p 3 CC S H

C Cd S l o

CQ tJ Cd

5

> o

cu >

0 CO o 0 cd o

w >

CO o

bO 0) B CO • H -Q H cu •p CO o

a 0) s u u s cu cu Si •p cu cd EH O H

CQ 0) •H •H CQ Cd u cd PL,

+ +

+ +

+ +

+

*

+ +

+ +

1

X SH a cd <D o H -p > •H CQ

•H -P O O o •p

c •H

-P Cd

s H g Q)

a Q) •H Cd bO 0) s II

cd

Q) •P Cd

<u O S

+ +

cu CQ C CD

+ + +

48

also been reported by Fujimoto and Adams (l964) that d i f f f e r e n t

collagen occur in the cut i c le and muscles of Ascaris. However,

there i s no conclusive evidence to understand the precise fun-

c t ional role of collagen in helminth parasites. It i s l i k e l y

that collagen f i b r e s may provide mechanical strength to the

t issue of the parasite , in order to f a c i l i t a t e active movement

inside the host, as according to Lehninger (1970) collagen of

vertebrates helps in binding a group of c e l l s together to form

a t i ssue . Furthermore, the collagens of d i f ferent chemical

configuration may be helpful to the parasite in d i f f erent ways,

as i f one type i s related with eggs, the other type might be

having some immionogenic properties. Further studies on the

collagen synthesis are however, required to ascertain i t s true

functional role in the trematodes.

Elastin i s another structural protein which i s intensely

present in the outer layer of the tegument and the caecae of

G. crumenifer. Moderate amounts are seen in the suckers, ven-

t r a l pouch, testes and parenchyma while other regions show

very weak staining (Table VI) . In P. epiclitum as well as in

G. explanatum, the distribution and intensity of staining i s

almost the same as in G, crumenifer. The eggs of G, explanatum

also show moderate staining f o r e las t in .

The ear l i er reports, though very few, suggest the presence

of e last in in the egg shel l of d i f ferent trematodes. Elastin

d i f f e r s from collagen not only in i t s chemical composition but

also in i t s so lub i l i ty in inorganic or organic solvents. For

H >

w

EH

^ CO P< <H S <H

B •S o

•H C -P •H n +3 0) cQ a cd o H -d

«H «H O O

CO fl cu O -P •H -H +3 to CO cd N i-t

•H Cd H ft

>

CO bO bO W

•H >

I 0) o w -p •5 S H

c cd CD e cd Ou o

CO X) cd C 5

> o

-p fi CD e a 0)

EH

Oi >

cu cd o <D Cd o

CO >

CO • o

bO 0) EH CO • H ^ H 0) CO o

OJ 0) O H

CO QJ •P •H CO Cd Jh Cd

PL.

49

+ +

+ +

+ +

+ +

+ +

+ +

+ + + +

i a w X JH s .J cd <1) o •H

H «H H > •rH CO O

•H •H P o a p CU (H S •P cd CO o cd cd C5 pL.

+ +

+ +

+ +

+ +

+ +

+ +

+ + +

cu H - p

o o o

•p

§ •H C5

H g (U

c 0) cu CO

•p

o s

OJ •p

cd bO cu s

I

0) •p cd <u Ti

+ +

<u w G cu •p

+ + +

50

instance collagen dissolves in 2% acet i c acid while e last in i s

remarkably insoluble (Pearse, 1968). Further, the e l a s t i c tisr-

sue i s known to be made up of 90? non polar aminoacids while

collagen has only 50? . It appears from the results obtained

in the present study that elastin i s widely distributed in

trematode body, part icularly in the region of high muscular

a c t i v i t y . It i s suggested that e last in may help the worms in

keeping the tone of the ir muscles and maintain e l a s t i c i t y to

recover the or iginal shape af ter the wave of contraction or

relaxation.

Among the structural proteins, f i b r i n i s another important

one, f o r i t s role in the motil ity of the parasite and patho-

physiology of the host. Distribution of f i b r in in the case of

G. crumenifer has been found intense in the suckers, caecae,

ventral pouch, parenchyma, i n t e r e s t i t i a l tissue and musculature.

Moderate staining i s also observed in the outer layer of the

tegument, gonads and lymph vesse ls . The same results heve been

obtained with P. eDiclitum and G. explanatum. The tes tes , vitel—

l a r i a , uterus and eggs of G. explanatum show intense to moderate

amount of f i b r in (Table VII ) .

Fibrin i s eos inophi l i c , homogeneous and usually f a i r l y

strongly PAS pos i t i ve . Its presence in the t issues out side the

blood vessels (in higher animals) i s associated with acute

inflammation and increased vascular permeability as a result of

damage to the vessel wall (McManus and Mowry,i960). Reports on

the general distr ibution of f i b r i n in trematode body organization

<D B o •p o CO •H

tH «H

CO f

C O H *H •H H •P > CQ

•H CU W U a o eq •H tj < H tH o O

cn c cu o •H •H -P CO CO CO N U •H CO H P-CO O o

CO

bO W

-P •H >

I 0 ^ h O K1 +3 3 cd

I c a o

fl 0) B a 01 eh

CQ 

o CO o (1) CO o

C O >

C O

o

bO OJ E H CQ • H -9 ^ (u CO o

IH j-i 0) cu •P

CO o H

+ + + + + +

* • + + +

+ * + +

+ + + +

+

c (U

+ + + Q) + + + CO + + +

-P O s

II + + + + + + * + + +

CD > •H

+ + + -P + + + CO

fio OJ s

+ + + + + + II

+ , + + * * +

CD

H

+ + + + + + + + +

CD •P CO

+ + + U + + + CD + + + •d

s

+ + + II + + + + + + +

+

(D CO C CD

+ + + + +

II

+ + + + + + +

s e X U i -p CO QJ o •H H M H >• •H CO o

a •H •H -P CD x! p O s P CD SH a

u CO CQ o u CO CO C5 cu

cu H >1 o o o § iaC •H O

51

52

are also not available and hence no generalization i s possible

unless further extensive studies are carried out. It may be

suggested that f i b r i n might be functioning in association with

co l lagen.

Tryptophan i s an important protein bound amino ac id - re la -

ted with the quinone tanning of the eggs of trematodes, ces to -

des and nematodes. Strangely enough i t does not occur in the

f r e e form in any of the three amphistomes under investigation,

as reported by Siddiqui (l984, unpublished). In the present

study, the outer layer of the tegument of G. crumenifer has

been observed to possess intense amount of tryptophan while

moderate intensity i s noted in the subte.gijimentary c e l l s , suck-

ers , lymph vessels and caecae. The other regions show weak

react ion. The distr ibution and loca l i zat ion of tryptophan in

the various regions of P. epiclitum has been the same as in

G. c rumen i f er but in lesser amoiants (Table VI I I ) . In the case

o f G. explanatum the outer tegumental layer did not show pos i -

t i ve reaction. Only caecae show intense presence while suck-

ers and gonads are found to have moderate intensity , the other

regions possess l i t t l e amount of tryptophan.

This amino acid was reported from P. westermani (von

Brand,1973) and also in the tegument, muscular and basal layers

o f the intestinal proteins of H. ovis (Soboleva and 2dafska,

1983). However, no functional role can be assigned to i t except

that i t may be involved in the stabi l izat ion of the egg-shel l

proteins or may be a integral component of other long chain

proteins . Further studies are however required to elaborate

H H

W a m << EH

0) s o •p to

• H O X ! H ft cd S

O C -H 3 -p ^ CO p. 

bO bO W P

• H >

I QJ ^ b O 13 to -p 5 OJ

S H

CO I c s i CO ^ CU O

CQ T) CO C

> o

-p c Q ) s ^ o

E H

>

0) CO o 

C O

o

tio CD Eh to ^^ Xi H P CD

C O O

u ! H 0 0 ) -p S CO o H

+ +

* *

a Q J

+ + + CD CQ

•P o

S

II + + +

* •k

CD > •H

* + + •R + CO

M 0)

S * + + S

+ II 1

+ * * I cu CD IS II

+ + + + + + +

+ 01 + 3

CO + + + JH + + + CD

•d o

S + + + II + + +

+ +

<D CQ C <D

+ + + -P +

II + + 1 + + + + + +

1 1 0) X J-. S -p H CO 0) o •H > H <H H

CQ •H CQ O O CD C •H > H O

• P Q ) Xi P O •H o B P <D - P CQ p s c CO ti CO CO F N CO o SH W CO CO CO • H o CU

cu

53

54

the nature of functioning of tryptophan in trematodes.

Nucleic acids are the most important biochemical cons t i -

tuents of the trematode body organization. The loca l i zat ion

of DNA and RNA i s almost similar in a l l the three species.

The main s i tes of intense to moderate presence of DNA are the

caecae, teguments and testes while RNA i s present in higher

amounts in the suckers, caecae, gonads and v i t e l l a r i a . The

lymph vessels of a l l the three species have been found to pos-

sess l i t t l e RNA, The ventral pouch of G. crumenifer has more

RNA and l i t t l e amount of DNA (Table IX).

The presence of nucleic acids in the caecae and i t s con-

tents indicate that they might be derived from the host in as-

soc iat ion with other substances. Further,.the ubiquitous pre -

sence of DNA and RNA in d i f ferent structures, part icular ly the

gonads, suggest that possibly these nucleic acids are playing

an important role in growth, reproduction and in protein syn-

thes is .

Most of the available data on the nucleic acids of para- '

s i t e s deals with the quantitative studies on various proto -

zoans, but very few helminth parasites , part icularly trematodes

and more so amphistomes have been investigated. Though there

are few studies on the d i f f e r e n t i a l distribution of nucleic

acids in the body organization of trematodes, they are in agre-

ement with the present study. For instance Smyth (l956) repor-

ted the presence of RNA in higher concentrations in the hold-

f a s t and gut c e l l s of C. bushinensis. However, great variations

OJ a o -p to •H ^ o & CO «H a •H

a ^ o •H •p

CQ t:) 0) a o -d § t-1 CQ < << S 'H H Q o <H CQ O CU -P S -H O to •H CO •¥> U CO CO N P, •H H CO O O

> 1-1 w t>0

•H >

E I to -p ^ <0 g H

C CO o

•p (1)

<D

ou >



CO

o

bO 0) H m • H H

CO o

tn fn 0) 0) O H

I + + + +

+ + * * * + +

+

* * * * + + + +

+ + + + + + + + + +

+

+ + + + + + G Q) + + + + + OJ

+ £Q -P O S II

+ + + + • + + + + + *

0) > •H

* * * * + + -P + CO bO (U

+ + + + + + a + + + + + + a + + + + H

i 1 + + + + * *

+ + CO o

+ + + + + + II + + + + + + + + + + + +

 H «H -p H -P 01 > •H CO O O Q) •H •H o -P +> CU Xi P o •H O s P CU m U 13 q CO -P ^ § a to O is h£ CO CO O CO •H PU o Oi o

55

56

have been found in the amount of DNA, depending on the develop-

ment of the v i te l lar ian c e l l s of F. hepatica (Govaert,1953).

Erasmus and Ohman (l963) found that the spindle shaped secre-

tory c e l l s associated with the adhesive organs of s t r ige id

trematodes possess higher amounts of protein and RNA thereby

suggesting that they are the s i tes of active protein synthesis.

According to von Brand (l979) nucleic acids in parasites occur

almost extensively as nucleoproteins. But i t has not yet been

established that though helminths can synthesize nucleic acids,

whether they can also synthesize purines and pyramidines de

novo or rely on those of the host or ig in (Smyth and Halton, 1983).

Therefore, the synthesis of nucleic acids in trematodes appears

to be a f i e l d which cal ls f o r further studies to f i n a l l y assess

and elaborate the b i o l og i ca l functions of this genetic material

which i s responsible f o r the storage, transmission and transla-

t i on of genetic information.

57

Histoenzymologlcal and Isoenzyme studies;

Earlier histochemical and electrophoretic studies on the

occurrence, distr ibution and isoenzyme pro f i l e of certain enzy-

mes of important metabolic pathways were made in a very few

trematode species (see l i terature in von Brand,1979; Chappell,

1980; Barrett,1981 and Smyth and Halton,1983). Furthermore,

no comprehensive data are available to compare with the d i f f e r -

ent trematode species inhabiting d i f ferent environmental condi-

t ions in the same host.

In recent years histoenzymological and e lectrophoret ic

techniques have proved to be useful t o o l s f o r the characteriza-

t ion of enzymes. In trematodes, since the iso lat ion of various

organs i s not poss ib le , the l o ca l i zat ion of enzymes in various

organ systems can only be possible by the histoenzymological

techniques which throw l ight on the metabolic a c t i v i t i e s of a

s p e c i f i c enzyme in a particular organ. Whereas, through the

e lectrophoret ic technique the possible existence of an enzyme

with i t s isoenzymes and their role during the developmental

physiology and transfer from one habitat/environment to another

can be studied. Further, the study of the isoenzyme p r o f i l e

a lso helps in the biochemical taxonomy of the worms.

58

The fol lowing results of the histochemical and e l e c t r o -

phoretic study of the enzymes of some amphistome parasites are

exclusively based on the above two techniques.

Glucose 6~phosphate dehydrogenase; (G6-PDH)

Histochemical;

This enzyme belongs to the pentose-phosphate pathway.

Intense act iv i ty of G6-PDH is present in the oral sucker and

caecae of G, crumenifer. The enzyme i s present moderately in

the inner layer of of the tegument, ventral sucker, gonads,

musculature and lymph vessels while the other regions show weak

enzyme act iv i ty . In P. eplclitum the ac t iv i ty of th i s enzyme

i s more intense in the ovary than other structure's which show

more or less similar distr ibution and intensity of staining as

in G. crumenifer. The distribution of G6-PDH in G. explanatum

i s not much d i f f erent from the other two species except that

the inner layer of the tegument i s weakly stained while caecae

and v i t e l l a r i a show intense enzyme ac t iv i ty (Table X; Plate I ,

Figs. 1 -3 ) .

Earlier Dodin £t a l , (l966) reported the presence of

G6-PDH in the subtegumental c e l l s of the males and in the

v i t e l l i n e glands of the females of S. mansoni. They found more

pronounced a c t i v i t y in the males than in the females. This

enzyme has also been reported from F. hepatica (Humiczewska,

1969). The present f indings on the d i f f e r e n t i a l distribution

of G6-PDH in the various structures of the three amphistomes

J>

>

m !aO tio w

-p D

•H >

I Q) o to S H

C cd gj s (0 O

> o CQ TJ CO a OE-i

O) >

cu CO o (D cd o

CO >

CO o

c (U a

eh

JH 0) Q) tt) !>. c ca •H H

f-l 0) 0) •p 3 cfl O H

+ +

+ + 59

+ +

+ +

+ +

+ + +

+ +

+ + +

+ +

+ +

+ + +

+ +

+ +

+ +

+ +

s -p cd 0) o •H H «H -p H CO >• •H CQ O (U G •H •H •P 0) XI P •H o s P Q) CO IH 3 Q) cd -P §

CQ O CO cd Cd £1. O cu

+ +

+ + +

+ +

+ +

+ +

+ + +

+ +

0) H > -P O o o 4-> §

bfl •H O

H

 •H a to CD s II

II +

Q) -P 03 S 0) rf

+ +

0) CQ C ou •p

+ + +

Plate VIII Electropherograms with corresponding densitometeric scans of polyacrylamide ge ls , stained f o r glucose-6-phosphate dehydrogenase ac t i v i ty . Numbers on the scans represent the bands. Arrows indicate the direct ion of enzyme migration.

Figures:

A. Gastrothylax crumenifer

B. Paramphistomum epiclitum

C. Buffalo rumen

D. Gigantocotvle explanatum

E. Buffalo l i ver

61

are in accordance with the ear l ier f indings on P. c e rv i ;

Plagiorchis elegans; C. michiganensis and Ganeo tigrinum

(Pat i l and Rodgi,l976b; Lef lore , I978j Leflore ejt ^ . , 1 9 8 0 and

Sharma and Mandawat,1983).

SlectroTPhoretic studvt

In the case of G. crumenifer four bands of isoenzymes

of G-6-PDH are detected. Isoenzymes 2-4 are cathodal in migra-

t i on while band 1 i s in the middle region of the gel (Plate

VII I , Fig, A). However, in P. epiclitum at least f i v e d ist inct

bands have been obtained. Band 1 i s anodal, band 2 i s in the

mid-gel region while 3-5 are cathodal bands (Plate VIII , Fig. B).

The host tissue buf falo rumen yielded only four isoenzyme f r a c -

t i ons o f G-5-PDH, Bands 1 and 2 are more towards the anodal r e -

gion, band 3 i s in the middle of the gel while band 4 migrates

towards the cathode (Plate VIII , Fig. C) .

The parasite of buffalo l i v e r , G. explanatum shows the

presence of four isoenzymes of G-6-PDH, Band 1 migrates towards

the anode, band 2 i s in the mid-gel region whereas bands 3 and

4 are more near the cathode (Plate VIII , Fig. D). In the host

t issue (biiffalo l i v e r ) only three isoenzymes of G-6-PDH have

appeared (Plate VIII , Fig. E) . Bands 1 and 2 are near the mid-

dle region of the gel while band 3 moves s l ight ly towards the

cathode.

Humiczewska ( l975a,b ,c ,d ) also reported the presence o f

G-6-PDH act iv i ty in the various developmental stages of

62

F. hepatica. and suggested the existence of pentose-phos-

phate pathway. Further, the d i f ferences have been noted by

several workers in the number and pattern of isoenzymes of

th i s enzyme between the larval stages and their respective

adult forms as well as between the adult worms of d i f f e rent

species and also among c lose ly related species (Coles,1970a;

Agatstoma and Suzuki, 1981; 1983; de Boissezon and Jelnes, 1982).

These authors have been of the view that isoenzyme studies

might help in quick ident i f i ca t i on of d i f ferent worms even

at the ir larval stages. In the present investigation cons i -

derable d i f ferences have been found to occur in the number

and electrophoretic mobility o f G-6-PDH isoenzymes among the

three amphistome parasites, two of them belonging to the

same habitat in the same host. For example, both G, crumeni-

f e r and P. epiclitiom parasit ize the rumen but the former

contains four isoenzymes while the l a t t e r exhibits f i v e . On

the other hand the isoenzymes of the parasite and the host

t i ssue d i f f e r considerably in the number and mobility as i s

the case with rumen and i t s parasites . Similarly the isoen-

zyme number of G. explanatum i s d i f f erent from P. epiclitum.

Though the number i s similar v/ith the isoenzymes of G. cru-

menifer but with d i f ferent mobi l i t ies . Moreover, the d i f -

ferences in the number and mobility i s also noted in G, exp-

lanatum and the host tissue ( l i v e r ) , the parasite possesses

four bands while host tissue shows only three bands of G-6-

PDH isoenzymes.

63

6-phosphogluconate dehydrogenase; (6-PGDH)

Histochemical studyt

This i s another important enzyme related with pentose-

phosphate pathway (shimt). The histochemical l o ca l i za t i on of

t h i s enzyme in G. crumenifer shows that the outer layer of the

tegument, caecae, ventral pouch and muscles possess moderate

a c t i v i t y . The highest enzyme ac t i v i ty i s noted in the suckers

while the other regions are weakly stained. The distr ibution

of t h i s enzyme in P. epiclitum i s a l i t t l e d i f ferent from G.

crumenifer in that the outer tegument and suckers show moderate

enzyme act iv i ty whereas caecae give higher intensity of stain-

ing (Table XI) . A t o t a l l y d i f f e rent pattern of distr ibution

of t h i s enzyme i s observed in G, explanatum. In th i s case the

outer layer of the tegument, v i t e l l a r i a and eggs are strongly

pos i t ive while the other regions except the lymph vesse ls

(which show l i t t l e enzyme ac t i v i ty ) have moderate presence of

t h i s enzyme (Plate I , Figs. 4 - 6 ) .

Previous histochemical observation by Humiczewska (l969)

revealed the presence of this enzyme only in the parenchyma of

F. hepatica while no act iv i ty was observed in the parenchyma

of D. lacteum. Pat i l and Rodgi (l976b)found that though the

enzyme was present in the oral sucker, sub-tegument, genital

cup, parenchyma surrounding the caeciAm and periphery of the

t e s t e s and ovary, the ac t iv i ty has been more pronounced in the

parenchyma surrounding the caecaum. The present f inding

o -p w •H

n •H

03 w cd

H T>

O bO «H O

!x;

s . eh

o XJ

o -p CQ 0) (U •P Cd

o O «H 3 o

e o T)

W CD •H CO CQ CO

ft CO « ft

«H o fi o •H N •H O o

> •J CO tiO WD W

-P

-P •H >

I (U o 3 CO -p p CO g H

fj CO C0X3 Pu O

CO -d CO c 5

> o

cu >

CO

64

+ +

+ +

+ +

+ +

+ +

+ + +

+ +

+ + +

+ +

+ +

+ +

O + + QJ + + CO + O • + + CO + + • +

>

• + + CO + + « + + o

Sh J-i OJ (U + + c; >> + + -p C CO + a •H H (U s (H p 0) > 1 + (D CO + EH O H 1 s (U

y u a •p H CCl • •H CQ O o 0) •H •H o +5 +> CU P o •H o s P CU -p CO tn s CO •p J-4 CO cO ^ CQ o w CO CO CO •H CL. o (U C5

a 13

 •H

tiO Q) S 11 I «

ti

<u -p CO u ID n

+ +

<D CO a 0) -p

+ + +

PLATE I .

F ig .1 . Gastrothvlax crumenifer. Frontal section showing glucose-6-phosphate dehydrogenase ac t iv i ty in the ventral sucker (VS) and parenchyma (P), X 400,

F ig ,2 , Paramphistomum epiclitum. Frontal section showing gluco se-5~pho sphate dehydrogenase ac t iv i ty in the oral sucker (OS), paren-chyma (P) and tegument (Teg). X 400,

Fig ,3 . Glgantocotyle explanatum. Frontal section showing glucose-6-phosphate dehydrogenase ac t iv i ty in the parenchyma (P), eggs ( E ) and testes (T), X 400,

Fig ,4 , Gastrothvlax crumenifer. Frontal section showing 6-phosphogluconate dehydrogenase ac t i v i ty in the oral sucker (OS), paren-chyma (P) and tegument (Teg), X 400,

Fig .5 . Paramphistomum epiclitum. Frontal section showing' 6-pho spho gluconate dehydrogenase ac t iv i ty in the parenchyma (P), tegument (Teg) and intest inal caeca ( i n t ) . X 400.

Fig ,6 . Gigantocotvle explanatum. Frontal section showing 5-pho spho gluconate dehydrogenase ac t i v i ty in the oral sucker (OS), lymph vessels (Lv) and tegument (Teg), X 400,

Plate IX Electropherograms with corresponding densitometeric scans of polyacrylamide ge l s , stained f o r 6-phosphogluconate dehydrogenase a c t i v i t y . Numbers on the scans represent the bands. Arrows indicate the direct ion of enzyme migration.

Figures:

A, Gastrothylax crumenifer

B, Paramphistomum epiclitum

C, Buffalo rumen

D, Gigantocotvle explanatum

E, Buffalo l i v e r

66

d i f f e r from that o f Hiiraiczewska (l969) with respect to the

r e s t r i c t i o n of this enzyme only in the parenchyma, but do not

d i f f e r s igni f i cant ly with the histochemical f indings of Pat i l

and Rodgi (1976b). As far as the presence of 6-PGDH i s con-

cerned i t has been reported even from the rediae and cercariae

hepatica (Hxjmiczewska, 1975c,d),

Electrophoretic study;

crumenifer possess at least f i v e isoenzymes of 6-PGDH

(Plate IX, Fig, A). The f i r s t two bands are s l ight ly towards

the anode. Band 3 i s in the middle of the gel while band 4

moves into the cathodic region and 5 i s nearest to the cathode.

Whereas in P. epiclitum also f ive d i s t inc t bands of this enzyme

are resolved. Isoenzyme l and 2 migrate towards the anode while

bands 3 and 4 are in the mid-gel region^ band 5 i s s l ight ly

cathodal (PlatelX, Pig, B). The rxjmen tissue shows the presence

o f four isoenzymes of 6-PGDH. Isoenzyme bands 1 and 2 are ano-

dal , band 3 i s in the middle region of the gel while band 4

migrates s l ight ly towards the cathode (Plate IX, Fig. C), Though

the number of isoenzymes of 6-PGDH i s similar in G. crumenifer

and P. epiclitiom. their mobi l i t ies d i f f e r considerably. Similar-

l y the number and posit ion of the isoenzymes of the host tissue

i s d i f f e rent from that of the above two parasites.

In G. explanatum, six isoenzymes of 6-PGDH are observed.

Isoenzyme l and 2 move towards the anode, the third i s in the

67

mid-gel region -whereas bands 4-6 migrate towards the cathode

region (Plate IX, Fig, D). The same enzyme has been found to

separate into seven fract ions in the case of l i v e r homogenate

(Plate IX, Fig. E) . The f i r s t three isoenzymes are anodal

in migration, bands 4 and 5 are in the middle of the gel where-

as bands 6 and 7 have s l ight ly cathodic af f inity .Here again,

there i s no s imilarity in the number and pattern of the isoen-

zymes of G, eyplanatum and the other two amphistomes p a r a s i t i -

zing rumen. Furthermore, the host t issue ( l i ver ) isoenzymes

not only d i f f e r in number but also in their e lectrophoretic

pattern, i f compared with the number and pattern of the isoen-

zymes of i t s parasite G. explanatum.

Agatsuma and Suzuki ( l98 l ) reported that isoenzyme pro -

f i l e of 6-PGDH of P. ohirai and P. miyazaki were considerably

d i f f e r e n t . They not only found the di f ferences between the

two parasites but also between the parasite and the host.

Similar results have been obtained by Agatstmia and Suzuki (1983)

in the case of S. mansoni and S. .japonicum and their host t i s -

sue, (mouse blood and muscle). These previous studies are in

t o t a l agreement with the present study, as far as the variation

in the number and electrophoretic mobility of the isoenzymes

among the three amphistomes as well as their respective host

t i s sues are concerned.

Malate dehydrogenase; (MDH)

Histochemical study;

Malate dehydrogenase i s an important TCA cycle enzyme

68

which has been found to occur in almost a l l the helminth para-

s i t e s studied to date. The loca l i zat ion and d i f f e r e n t i a l d i s -

tr ibution of th is enzyme in the three amphistome species i s

summarized in Table XIII . It has been found to appear modera-

t e l y in the suckers, ventral pouch, gonads, parenchyma and

muscles of G, c rumen i f er while the other regions possess weak

a c t i v i t y and the outer tegumental layer i s completely negative.

In the case of P. epiclitum, a parasite l iv ing in the same ha-

b i t a t , the d i f f e r e n t i a l distribution of MDH act iv i ty i s similar

to G. crumenifer except that the caecae and inner tegumental

layer show intense and moderate enzyme ac t iv i ty , respect ively .

The overall ac t iv i ty of this enzyme in G. explanatum appears

to be much higher than the other two species. Here also the

outer tegument has been found negative whereas the inner tegu-

ment and oral sucker show high enzyme ac t i v i ty . The other re -

gions except the eggs show moderate ac t iv i ty of MDH (Plate X I I ,

Figs. 1 -3 ) .

The ac t iv i ty of MDH has been reported in most tissue of

Pharyngostomoides adenocephala (Allen,1972). This has been

found to occur even in the developing sporocyst, rediae, c e r -

cariae and metacercariae of F. hepatica by Humiczewska (l975a,

b , c , d ) . Almost similar observations have been made by Leflore

( l978) and Leflore et (l980) in the case of P. elegans

and G. jnichiganensls respect ively .

H H

3

0) s o -p w •H

cd

to c« C 0) bO o X T)

o «H <D «H

C •H

O •H -P to (U e o . . <1> tJ

pq "d < <H H CU o 4J 03 CO

H 0) CO -p S fi

CO CO

O CO

c o •H +> cd N - 1 •ri H CO o o

>

W bO bO W

4J •H >

I 0 o m -p 3 cc S H

C CO 0) S CO ^ Oi O

CQ TJ CO c 5

> O

c CU a s 0) EH

CIi >



CO

o

f-i u CU (U fi ^ C CO •H H

t. U 0 (U 4-> >> 3 CO O H

01 Q) •H tQ CO

CO CU

+ + 69

+ +

+ +

+ +

+ +

+ +

+ +

+ +

H

-P 0 iU 01 CO O

+ +

+ +

+ +

+ + +

+ +

+ +

+ +

+ +

+ +

+ +

+ +

+ +

+ +

+ +

+ +

+ + +

+ + +

e i Q) S -p H o >1 H CO O O •H •H O X! P O p CU -P a C CO CO w CO •H a, O

-P CO

0)

c Qi d) 01

o 3

CU >

•H W) 0) a n

ti +

(D -P CO u Q) -d

+ +

<u to a 0)

+ + +

Plate X Electropherograms with corresponding densitometeric scans of polyacrylamide ge l s , stained f o r malate dehydrogenase a c t i v i t y . Numbers on the scans represent the bands. Arrows indicate the direct ion o f enzyme migration.

Figures:

A, Gastrothylax crumenifer

B, Paramphistomum epiclitum

C, Buffalo rumen

D, Gigantocotyle explanatum

E, Buffalo l i v e r

71

Slectrophoret ic study t

In G, crumenifer there are atleast seven isoenzyme bands

of MDH, Two isoenzymes (l & 2) are anodal whereas 3 and 4

are in the mid-gel region (Plate X, Fig, A). Bands 5-7 migrate

towards the cathode,In the case of P, epiclitum the number of

isoenzymes is the same as in G. crumenifer but with d i s t inc t ly

d i f f e r e n t mobi l i t ies (Plate X, Fig. B). Isoenzymes 1-3 are

anodal while the last four bands 4-7 are cathodal, 6 and

7 being nearest to the cathode.However the host tissue rumen

shows only four MDH isoenzymes, which i s d i f ferent from the

number of isoenzymes obtained in the case of i t s two parasites .

S igni f i cant ly enough there i s no isoenzyme band in the mid-gel

region and bands 1 and 2 are anodal whereas bands 3 and 4 are

near the cathode (Plate X, Fig, C).

In G. explanatum there are four isoenzyme bands of MDH

(Plate X, Fig. D), The number and pattern of isoenzymes are

quite d i f ferent from the other two amphistomes of rumen. I s o -

enzyme bands 1 and 2 are anodal while band 3 i s near the mid-

dle of the gel and the fourth band moves towards the cathode.

The isoenzyme number of the host t issue ( l iver ) i s seven. The

bands are not only more in number but also d i f f e r in the ir mo-

b i l i t i e s from the isoenzymes of the parasite, G. explanatum.

Though the number of isoenzymes of MDH in l i v e r i s similar to

the other two parasites, i , e , , G, crumenifer and P, epiclitum

but in th is case also the mobi l i t ies are d i f f e rent . Band 1 i s

72

most anodal, band 2 i s s l ight ly towards the anode, band 3

i s in the mid-gel region whereas isoenzymes 4-7 show cathodlc

mobil ity (Plate X, Fig. E).

In an e a r l i e r study, 2 MDH isoenzymes have been reported

from adult and cercariae of S, mansoni by Conde-Del Pino et a l .

( l966) and Homewood et ad. ( l973) . But in another study Oya et

a l . (l970) found that adult S. mansoni possesses 4 MDH isoenzy-

mes whereas there have been only three in adult S. .japonicum.

They however did not f ind any d i f ferences in the isoenzyme pat -

terns between d i f f erent sexes of th is species. Inter and intra

s p e c i f i c variation in the number as well as pattern of MDH i s o -

enzymes have been observed in S. mansoni (Coles, 197Tb). Yan et

(l976) also reported varying numbers of MDH isoenzymes in

d i f f erent strains of S. .japonicum and sub-species of Oncomelania

hupensis. As far as MDH isoenzymes of d i f ferent strains of

Schistosoma spp., are concerned, varying numbers have been repo-

rted by d i f ferent workers (Ross,1976; Ross et a l . , l 9 7 8 ; Rotmans,

1978; Southgate £ t ^ . , l 9 8 l ) . It has been noted by most

previous workers that the study of MDH isoenzymes may be s igni -

f i cant f o r the d i f ferenc iat ion of a given species at intra-spe-

c i f i c l e v e l . The d i f ferent number and pattern of MDH isoenzy-

mes, among parasites as well as between parasites and their

host tissue observed in the present investigation are therefore,

in agreement with ear l i e r f indings. An interesting study by

Dhandayuthapani et (l983) that G, crumenifer obtained from

three d i f ferent hosts (buf falo , goat and sheep) shows quite

73

s ign i f i cant variat ions in the number of MDH isoenzymes. Hence

they suggested that the variations may be due to the influence

o f the host. But as far as the number of MDH isoenzymes in

c rumen i f er are concerned the present study i s not in agree-

ment with the above f indings. They have reported two isoenzy-

mes while presently, seven isoenzymes have been foun<Jin G. cru-

menifer from buf fa lo rumen. Though the number of isoenzymes

of G. crumenifer described in the two studies i s considerably

d i f f e r e n t but i t i s d i f f i c u l t to understand as to what factors

are responsible f o r th is d i f f erenc iat ion . Whether th is i s due

to the d i f ferent strains/species of the parasite as well as of

the host; dietary e f f e c t s of the host or di f ference in the e lec -

trophoretic conditions l ike gel concentration, molarity and pH

of the buffers might have been some of the influencing fac tors .

Succinate dehydrogenase;(SDH)

Histochemlcal study:

Succinate dehydrogenase i s another TCA cycle enzyme which

i s also c lose ly connected to the respiratory chain of parasites.

The distribution of SDH act iv i ty as evident from the intensity

of staining i s very much similar in a l l the three species under

study. The main s i tes of high ac t iv i ty are the caecae, oral

suckers and tes tes . Ventral pouch and ovary of G, crumenifer

also show intense ac t iv i ty of this enzyme. Moderate act iv i ty

has been observed in the inner tegumental layer, ventral suck-

ers , parenchyma and v i t e l l a r i a whereas weak ac t iv i ty i s seen in

0) S o 4J (Q U4

n -H o 

w no tJO w

4-'

•H >

I o

EH

PL. >

Si cO o +

• CO + + + • + + + o + + +

<D 0) + + + C + + + C CO + •H H H «H 4J H -P cd CQ > •H to o o c CI) .a G •r-i •rH o 3 +» •P CD xi o H •H o S p CU -p B to tl p X cd •p cO cd CU fH to o w a CO CO •H cu o CU C5

c 0) 0) Cfi +3 o a-

(U !> • H

•s DO OJ a

II +

0) CO 0) ts

+ +

<t> CO -P

+ + +

PLATE I I . Fig.1.

Fig .2 .

Fig .3 .

Fig4.

Fig .5 .

Fig .6 .

Gastrothvlax crumenifer. Frontal section showing l oca l i zat ion of malate dehydro-genase ac t iv i ty in the testes (T), paren-chyma (P) and ventral pouch (Vp). X 400,

Paramphistomum epiclitiom. Frontal section showing malate dehydrogenase act iv i ty in the ventral sucker (VS), parenchyma (P), tegument (Teg) and intest inal caeca ( i n t ) , X 400.

Gigantocotvle explanatimi. Frontal section showing malate dehydrogenase act iv i ty in the oral sucker (OS) and tegument (Teg). X 400.

Gastrothylax crumenifer. Frontal section showing (succinate dehydrogenase act iv i ty in the tegument (Teg), parenchyma (P). ventral pouch (Vp) and testes (T). X 400. Paramphistomum epiclitum. Frontal section showing succinate dehydrogenase act iv i ty in the oral sucker (OS), intest inal caeca ( In t ) and tegument (Teg). X 400.

Gigantocotyle explanatum.. Frontal section showing succinate dehydrogenase act iv i ty in the tegument (Teg), t es tes (T) and eggs (E). X 400.

aaATi oiBjjng

imq.BUBictxe axAr -Ooornrei jf) 'a

uaumJ oiBjjng •q

umq-TioTcia ainmo;.Sf:qaitrej:B<j

jajfuaumao XBXAitq.oan.SBo "y rsaoniTJ

•uox -BJ THi ainAzua j o uoxôsJixp aq-isoTpuT sMCuav 'spireq ^.ukaadaa suôs a^q. uo saaqumN •Aq.jAiq.oB

asBuaSoapAqap aq-Buxoons ao j pauTBq.s 's ia3 apTuiBxîoBiClod JO suBos OTaaâmorV suap

SuTpuods^-JJoo smBj:9oj:at{doj::}.oaxa JX

75

3

A

76

the outer layer of the tegument, uterus and lymph vesse ls .

The outer and inner layer of the tegument of G, explanatum

possess moderate and intense enzyme act iv i ty respect ively ,

which i s s l ight ly d i f ferent from the other two species (Table

XII; Plate I I , Figs. 4 - 6 ) .

The histochemical occurrence of SDH has been reported

also from the free l i v ing larval stages of F. hepatica. P.

elegans and C. mlchiganensis (Humiczewska 1975a,Leflore,1978;

Lef lore et ^ . , 1 9 8 0 ) . Among the mature worms F, hepatica. D.

lacteum, P. adenocephala, C. scoliocoelium and G, crumenifer

have been examined histochemically by Thorpe ( l967) , Humiczew-

ska (1969; 1975a),Allen (l972), Sharma (l977; 1978; 1979),

Gorchilova (l980) and Humiczewska ( l982) . The d i f f e r e n t i a l d i s -

tr ibut ion of this enzyme, as described by these ear l i e r authors,

almost coincides with the present study on the l o ca l i zat ion of

SDH ac t iv i ty in the three amphistomes of Indian water buf fa lo .


The electrophoretic analysis of SDH reveals the presence

o f three isoenzymes in G. crumenifer. Band 1 i s in the mid-gel

region while band 2 and 3 move towards cathode (Plate XI, Fig.

a ) . In P. epiclitum SDH i s separated into f ive isoenzyme bands.

Bands 1 and 2 are anodal, 3 and 4 are in the mid-gel region

whereas band 5 has s l ight ly cathodal mobility (Plate XI, Fig. B)

There i s a considerable di f ference not only in number but also

in the pattern of SDH isoenzymes between the two parasites

77

inhabiting buffalo rumen. In the case of the host t issue

(rumen) there are three d ist inct isoenzymes. Band 1 ie ano-

dal in migration whereas band 2 i s in the mid-gel region and

band 3 moves towards cathode (Plate XI, Fig. C), Though the

number of SDH isoenzymes of rumen i s similar to the number

found in G, crumenifer but their mobi l i t ies are quite d i f f e rent .

The number and pattern of isoenzymes of P. epiclitum and that

o f the host tissue are also d i f f e rent .

The l i v e r parasite G. explanatum shows four isoenzyme ba-

nds of SDH, Band 1 i s anodal band 2 i s s l ight ly towards the

anode whereas bands 3 and 4 move towards cathode (Plate XI,

Fig. D). The number and relative mobility of SDH isoenzymes of

explanatum d i f f e r from that of G. crumenifer and P. epiclitum.

belonging to the same host but parasit iz ing d i f ferent organs.

Marked di f ferences have been noted between the SDH iscSenzymes

of the host tissue l i v e r and i t s parasite G. explanatum. Only

three bands have appeared in the case of l i v e r . Band 1 i s an-

odal while bands 2 and 3 are in the mid-gel region (Plate XI,

Fig. E) . Interestingly the isoenzyme number of both the host

t i ssues (rumen and l i v e r ) i s similar but here again they are

d i f f e rent in their relat ive mobi l i t ies . This may be due to the

d i f ference in the biochemical nature of the two organs. However,

there i s no other e a r l i e r report on the SDH isoenzymes of trema-

tode parasites and the ir host.

78

cK-glycerophosphate dehydrogenase; (o<-GPDH)

Histochemical study:

c<.glycerophosphate dehydrogenase i s linked with the

sequence of reactions taking place in the g lyco ly t i c pathway.

This mitochondrial-bound f lavoprotein enzyme i s the other half

o f the "^glycerophosphate shuttle. It oxidises L -g lycero l -3 -

phosphate to dihydroxyacetone phosphate.

Histochemical l oca l izat ion of this enzyme shows uniform

distr ibut ion in d i f f e rent regions of G, crumenifer, P. e p i c l i -

tum and G. explanatum. The outer layer of the tegument of a l l

the three species give negative staining f o r o GPDH (Table XIV;

Plate I I I , Figs. 1 - 3 ) . There i s no s igni f icant variation in

the intensity of staining among the three amphistome species

but i t appears that G. crumenifer has a l i t t l e higher amount of

«»<-GPDH than the other two species.

There i s only one ear l ier report on the histochemical d i s -

tr ibut ion of this enzyme in adult worms by Pati l and Rodgi

(l976b)on P. cerv i . which i s almost in agreement with the pre -

sent f indings. However, «<-GPDH i s widely distributed in d i f -

ferent organs of larval forms as reported by several workers.

Humiczewska (l975c,d) reported the presence and distr ibution of

t h i s enzyme in the rediae, cercariae and metacercariae of F.

hepatica. Furthermore, Leflore (1978) found^GPDH in the tail^

tegument, caudal pocket, excretory bladder, acetabulum and oral

sucker, particularly in the muscles surrounding the sty let of

> H ><! W pq <j!

0) a o +» CO •H &

CQ cd C OJ . . tiO CO O <H f-4 tH X) P 0) o T) -H -P Q) CO -P 0) Cti S jc! o CO «H o ft o 0 O A' bO

«H O a o

• H

N • H

O O

o H

CO 0) •p •H CO CO fn CO P-

>

CO (50 bO M

-P •H >

i CU ^ !h o CO -p CO

C CO 0) e CO ^ CI. O

> C0|o •d CO cS EH

QJ CO o QJ CO o

CO >

CO

o

CU 0 C ^

- p C CO •H H Q) s fn f^

CU 0 5c - P >> o P CO

O H

+ + + •

* * + + +

+ * +

+ + + +

c CU 0 w + + + + + 4-' O S

II *

+ + + +

> •H •P CO + + + fciO + + + 0 + s

II + + + + + + 1 + +

CO + 0 + * * + II

+ + + + + + + 0 + + + -p CO

0 XJ + + + o + + + s + + II

+ + + + + + + +

+ + 0 CO c 0 •p + + + c + + + H

II + 1 1 1 + +

a s ^ 0 X a •p H p CO 0 o •H > -p H «H H -t-> CO CO >• •H CO O O Jill 0 G •H •H O ro -p -P 0 X! Pi o H •H o S p 0 P CO ;3 0 c X CO +> !H § CO 0 f-l CO o fn u as CO CO •H a, C3 CU a

79

Plate XII Blectropherograms with corresponding densitometeric scans of polyacrylamide ge ls , stained for-K-glycerophosphate dehydrogenase a c t i v i t y . Nxjmtiers on the scans represent the bands. Arrows indicate the direct ion of enzyme migration.

Figxires;

A. Gastrothylajc crumenifer


C. Buffalo rxjraen

D. Gig:antocotyle explanatum

E. Buffalo l i v e r

80

B

2 3

81

the cercarial stage of P. elegans. The cercariae of C. michi-

^anensis have also been found to possess the ac t iv i ty of this

enzyme in the d i f f e rent body regions (Leflore et a l , . 1 Q 8 O ) .

Electrophoretic study:

In the case of G, crxjmenifer. four molecular forms of

^GPDH have been obtained. Isoenzyme band 1 and 2 move s l i gh -

tly^ towards the anode whereas bands 3 and 4 have cathodal mig-

ration (Plate XII, Fig. A), In P. epiclitum this enzyme i s in

the form of f i ve isoenzymes, where band 1 i s more anodal, bands

2 and 3 are almost in the mid-gel region while bands k and 5

move a l i t t l e towards the cathode (Plate XII, Fig. B). The

number and relat ive mobil it ies of the isoenzymes of«<-GPDH of

G, crumenifer and P. epiclitum are d i f ferent as the former pos-

sesses only three isoenzymes while the la t ter has f i v e . The

host tissue rumen, however, possesses three isoenzymes of<-GPDH.

The f i r s t two bands (l & 2) are anodal in migration while the

third i s in the middle of the gel (Plate XII, Fig. C). On com-

parison, considerable di f ferences have been noted in the number

and pattern of the isoenzymes of the host tissue and that of

i t s parasites.

The third parasite, G. explanatum he s f ive d is t inct bands

of«>^GPDH isoenzymes (Plate XII, Fig, D). Bands 1-3 migrate

towards the anode, band 1 being most anodal, viiereas bands 4

and 5 are in the mid-gel region. There i s no similarity in

the-<S-GPDH isoenzymes of the three parasites except that the

number of the isoenzymes of P.epiclitx^m i s similar to G.

82

explanatum. However, the isoenzymes in the case of l i v e r are

four in number. Band 1 i s most anodal, band 2 i s in the mid-

dle of the gel whereas bands 3 and 4 migrate towards the cathode

(Plate XII, Fig, e ) . Here again the di f ference can be seen v e -

ry c lear ly in the number and pattern of migration of isoenzyme

bands of l i ver and that of G. explanatum. The two host tissues

(rumen and l i v e r ) also show dissimilarity in the number and mo-

b i l i t y of «<-GPDH isoenzymes. No ear l i er report on-K-GPDH i s o -

enzymes of trematodes i s available.

Hexose-6-dehydrogenase; (H-5-DH)

Histochemical study:

This enzyme i s also indirect ly related v/ith the main g ly -

c o l y t i c pathway and i s a part of intermediary metabolism. His-

tochemical l o ca l i zat ion of th is enzyme in the three amphistomes

revealed that the outer layer of the tegument of a l l the three

species i s devoid of enzyme a c t i v i t y . The oral sucker of G.

crumenifer i s intensely posit ive while ventral pouch shows nega-

t i v e reaction. The testes of P. epiclitum and the testes and

ovary of G. explanatiom possess higher amount of H-6-DH whereas,

the other regions give moderate to weak reaction (Table XV;

Plate I I I , Figs. 4 - 6 ) .


The isoenzymes of H-6-DH in the case of G. crumenifer are

4 in number. All the four fract ions migrate towards the cathode

i o -H •H Si PU

c o •H H CO 0) W «H B G 0) bO o o -H

Sh ^ -a u) <D M ^ a t-3 o m ts c • EH <H o o CO CO o CD X 4-» 

I 0 3 U O 3 to -p 3 CO S H

c eg 0 s b >» CO Xi CU O

CO o n CO fl Q •

• a. •

>

CD cd o <D CO o • CO •

>

• CO • o

u u d) 0) c ^ •p a CO a •H H OJ s p J-4 s p OJ 0) u •p Q) P CO O H

CO <D 4-' •ft CO CO Ih CO o.

+ +

+ +

+ + +

+ +

+ + +

+

O Fh -P CO CO o

+ +

+ + •f

+ +

+ +

a s B CU o •H H •H CO o c •H •H cu xi p

§ p <S) § o tl CO cu

+ +

+ +

83

+ +

+ + +

+ + +

+ +

d) H >• -P O o o

wl •H a

-p CO

H (U

PLATE I I I .

Fig . 1, Gastrothylax crumenifer. Frontal section showingo^glycerophosphate dehydrogenase ac t iv i ty in the tegument (Teg;, ventral pouch (Vp), ventral sucker (VS) and lymph vessels (Lv). X 400.

Fig. 2. Paramphistomum epiclitvim. Frontal section showingo^glycerophosphate dehydrogenase ac t iv i ty in the tegument (Teg), ventral sucker (VS), testes (T) and intest inal caeca ( I n t ) . X 400.

Fig, 3 . Gigantocotyle explanatxam. Frontal section showingo^glyc eropho sphate dehydro genase ac t iv i ty in the tegument (Teg), intest inal caeca ( Int ) and eggs (E). X 400.

Fig. 4 . Gastrothylax c r m e n i f e r . Frontal section showing hexose-5-dehydrogenase act iv i ty in the tegument (Teg), ventral sucker (VS) and lymph vessels (Lv). X 400

Fig. 5. Paramphistomum epiclitum. Frontal section showing hexose-dehydrogenase act iv i ty in the tegument (Teg), intest inal caeca ( i n t ) , testes (T) and lymph vessels (Lv). X 400.

Fig. 6 . Gigantocotyle e^lanatum. Frontal section showing hexose-5-dehydrogenase act iv i ty in the testes (T) , eggs (£) and lymph vessels (Lv). X 400.

J-V •Teg . /

Plate XIII Electropherograms with corresponding densitometerlc scans o f polyacrylamide ge l s , stained for hexose-6-dehydrogenase a c t i v i t y . Numbers on the scans represent the bands. Arrows indicate the direct ion o f enzyme migration.

Figures:



C. Buffalo rvmen

D. Gigantocotyle explanatum

E. Buffalo l i ve r

84

D

3 4

85

(Plate XIII, Fig. A). In P. epiclltum six isoenzymes were

observed; bands 1 and 2 are anodal whereas bands 3-6 migrate in

the cathodal direct ion (Plate XIII , Fig. B). Notable variation

in the number and pattern of the isoenzymes of the above two

parasites have been observed. Furthermore, the rumen shows the

presence of seven f rac t ions of H-6-DH, All the seven bands move

towards the cathode (Plate XIII, Fig. C), On comparison, con-

siderable di f ferences have been noted in the number and p o s i -

t i on of the host t issue isoenzymes and that of i t s two parasi -

t e s .

The l i v e r parasite , G. explanatum has been found to pos-

sess H-6-DH in the form of seven isoenzymes. Bands 1 and 2 are

anodal, band 3 i s s l ight ly in the middle of the gel whereas

bands 4-7 are of cathodic a f f i n i t y (Plate XIII, Fig. D). While

in l i v e r , six isoenzymes of H-6-DH are found. Bands 1 and 2 are

anodal while the other four bands (3-6) are cathodal bands

(Plate XIII, Fig. E). I t i s c lear ly seen that the niimber and

pattern of H-6-DH isoenzymes of the three amphistome parasites

i s d i f f e rent and also they do not have any s imilarity with their

respective host t i ssues . There i s no other report e ither on

the histochemical l o ca l i zat ion or on any electrophoretic study

on the isoenzymes of H-6-DH,

Fructose i.6-diphosphatase: (Fr1,6-DP)


This hydrolytic enzyme act ively part ic ipates in a by-pass

to the main g lyco ly t i c pathway sequence and helps in the f o r -

86

mation of glucose from l a c t i c or puruvic acid. This enzyme

gives weakly pos i t ive reaction in outer layer of the tegument

o f a l l the three species under study. Highest enzyme act iv i ty

i s observed in the intest inal caecae, oral sucker, gland c e l l s

o f ventral sucker, ventral pouch and gonads of G. crumenifer*

The other regions show moderate amount of this enzyme while

lymph vessels are weakly stained. Similar distr ibution and

intens i ty of th is enzyme i s seen in P. epiclitum except that

the gland c e l l s of the ventral sucker show moderate ac t iv i ty

(Table XVI; Plate IV, Figs. 1 -3 ) . In the case of G, explanatum

intense act iv i ty of Fr1,6-DP i s seen in the inner layer of the

tegument, oral sucker, gonads, v i t e l l a r i a and eggs, whereas the

lymph vessels show moderate a c t i v i ty .


In G. crumenifer. Fr1,6-DP i s in the form of nine isoen-

zymes (Plate XIV, Fig. A). Bands 1-3 are anodal, band 4 i s

near the middle of the gel while the other f i ve bands (5-9) are

cathodal in mobil ity. Only three isoenzyme bands of th is enzy-

me are found in P. epiclitum. Band 1 i s in the mid-gel region

whereas bands 2 and 3 move towards the cathode (Plate XIV,Fig.B).

Among these two parasites the former has as much as nine isoen-

zymes of Fr1,6-DP while the la t ter possesses only three and also

the migartion pattern of the bands i s dissimilar. But the rumen

t issue shows the presence of nine isoenzymes of this enzyme

(Plate XIV, Fig, C) . Isoenzymes 1-4 are anodal, band 5 i s in

M

M l-l PQ < EH

Tf tt>

> J (0 bO hO W

•P •H I> I (U ^ b O JJ CO ^ OS S H I C rt o

H

>



CO o

U U Q) QJ C >> +5 C CO c •H H > 0) D CO EH O H

+ + + +

* * + + +

• * +

+ + + + +

+ c OJ o m

+ + + + + + •4-> o s

If *

+ + + + + + 0)

> •H + + + CO + + + bO + + + OJ S + + + II + + + + + + 1

+ 0) + * * + It

+

+ + + 0) + + + -p + + CO

CU •d + + + £ + + + + u

+ + + + + + + + + + + •k • H «H •p H •P 0} >- •H CO O O 0} X! P o •H O g p (U -p CO fn P e q CO ^ CO 3 CO O ^ ti£ CO CO CO •H CU C5 CU o

87

Plate XIV Electropherograms with corresponding densitometeric scans of polyacrylamide ge l s , stained for fructose 1,6-diphosphatase a c t i v i t y . Numbers on the scans represent the bands. Arrows indicate the direct ion o f enzyme migration.

Figures:



C. Buffalo rumen



88

B

D

89

the middle of the gel while bands 6 -9 move towards the cathode

Though the number of bands in the case of G. crumenifer and the

host tissue are similar but they d i f f e r in their re lat ive mobi-

l i t y . However the isoenzymes of P. epiclitum are much l ess ,

(only three) in number than either the host tissue isoenzymes

or the isoenzymes of G. crumenifer»

In the case of G. explanatum also the number of Fr1,6-DP

isoenzymes i s nine (Plate XIV, Fig. D). Isoenzyme bands 1-5

are anodal^ band 6 i s in the middle of the gel whereas bands

7 - 9 migrate towards tho cathode.In the related host tissue

( l i v e r ) , seven isoenzymes of this enzyme have been obtained.

Bands 1-4 move towards the anode, band 5 is near the mid-gel

region while bands 6 and 7 have cathodal mobility (Plate XIV,

Fig. E) . The mobility of band 1 i s more or less similar in a l l

the cases except P. epiclitum in which the f i r s t band i s in the

middle of the gel . Otherwise there are considerable variations

in the number and mobility of Fr1,6-DP isoenzymes among a l l the

three species of amphistomes as well as with their respective

host t issues ,

Fr1,6-DP has ear l i e r been reported from F. hepatica (von

Brand,1979). However there is no other report available on the

histochemical l oca l i zat ion as well as isoenzyme study of this

enzyme in helminths except that Fr1,6-DP was par t ia l l y pur i f ied

by Kohler and Hanselman (l973) from D. dendriticum.

90

Glvceraldehvde-•3~phosphate dehydrogenase: (G-3-PDH)

Histochemlcal study:

This enzyme belongs to the g lyco lyt i c pathway and brings

about oxidation of glyceraldehyde-3-phosphate to y ie ld energy.

Histochemical e f f o r t s to l o ca l i ze G-3-PDH revealed that there

i s no enzyme ac t iv i ty in the outer layer of the tegument of a l l

the three species under study. Ventral sucker and ventral pouch

o f G, crumenifer also shoe negative reaction. Moderate enzyme

a c t i v i t y i s observed in the gonads of P. epiclitum while the

gonads of G, explanatijm possess high act iv i ty of th is enzyme

(Plate IV, Figs. 4-6 ; .Table XVII).

Humiczewska ( l975b,c ,d) reported the presence o f G-3-PDH

in the sporocyst, rediae and cercariae of F. hepatica. which

l ed him to conclude that g lyco lys is may be act ively providing

energy to- the above larval forms. Furthermore, th is enzyme has

been found to have high ac t iv i ty in helminth parasites (Barrett,

I 9 8 l ) , However, this enzyme has not ear l ier been l oca l i zed histo-

chemically in any adiiLt trematode parasite.


In the homogenate o f G. crumenifer. f ive bands of G-3-PDH

isoenzymes are seen (Plate XV, Fig. A) . All the bands show cath-

odal migration. In P. epiclitum six f ract ions of th i s enzyme

have been foijnd (Plate XV, Fig. B). Band 1 has anodic a f f i n i t y

whereas the other f i v e bands (2-6) have migrated towards the

a o m P.

> (D X! 0) •d x) M H Cd CO JH ^ CO (U ft o >> H fciO tH O c o •H -P CO N •ri H CO o o

to bO bO W

£ •p •H >

I 0)

O 3 to -P cd S H

I C CO , axi a, o

w c 5

> o

4-> C (U B Gi 



o

 •p •p H -p a •H to O o c •H •H o 3 CU X! P o H s P O -p p 3 a a X CO CO o o is bC o CO •H O

G 0) CO O

Q) > •H 4-' Cd bO CU S 11

II +

<D -P CO Jh <U TJ o s

+ +

0) to c CU

+ + +

PLATE IV.

Fig .1 . Gastrothylax crvwienifer. Frontal section showing fructosel,5-diphosphatase act iv i ty in the tegument (Teg), oral sucker (OS), parenchyma (P) and ventral pouch (Vp). X AOO.

Fig ,2 . Paramphistomum epiclitum. Frontal section showing fructose1,6-diphosphatase act iv i ty in the tegument (Teg), ventral sucker (VSj, parenchyma (P) and intest inal caeca ( I n t ) . X 400.

F ig .3 . Gigantocotyle explanatum. Frontal section showing fructose 1,5-diphosphatase act iv i ty in the oral sucker (OS), tegument (Teg) and eggs (E). X 400.

Fig .4 . Gastrothylax crumenifer. Frontal section showing glyceraldehyde-3-phosphate dehy-drogenase ac t iv i ty in the tegument (Teg), ventral pouch (Vp"), intest inal caeca (Int) and parenchyma (P), X 400.

Fig .5 . Paramphistomum epiclitum. Frontal section s ho wing glyce r aldehyde-3-pho sphate dehy-drogenase ac t iv i ty in the tegument (Teg)-, intest inal caeca (Int) and parenchyma (P). X 400.

F ig .6 . Gigantocotyle explanatum. Frontal section showing glyceraldehyde-3-pho sphate dehy-drogenase ac t iv i ty in the tegument (Teg), v i t e l l a r i a (V i t ) , parencyma CP) and eggs (E). X 400.

Plate XV Electropherograms with corresponding densitometeric scans of polyacrylamide ge l s , stained f o r glyceraldehyde-3-phosphate dehydrogenase ac t iv i ty . Numbers on the scans represent the bands. Arrows indicate the direction of enzyme migration.

Figures:



C. Buffalo rumen

D. Qigantocotvle explanatum


92

Q

3 4

1

93

cathode. The isoenzymes of G-3-PDH of the above two parasites

d i f f e r in number and mobility and also the major di f ference i s

the absence of any anodal band in the case of G. crumenifer

while i t i s present in P. epiclitum. The rumen i s fovind to

have seven G-3-PDH isoenzymes. Bands 1-3 are anodal while

bands 4-7 show cathodal migration (Plate XV, Fig. C) . Here

again the number and mobility of isoenzymes of the parasites

and the host tissue i s dissimilar.

explanatum contains only four isoenzymes of G-3-PDH

(Plate XV, Fig. D). band 1 i s anodal and bands 2-4 are catho-

da l . However, the l i v e r homogenate has been found to possess

s ix isoenzymes of G-3-PDH (Plate XV, Fig. E). Isoenzyme band

1 i s anodal, band 2 i s in the mid-gel region whereas bands 3-6

are cathodal bands. No similarity has been observed in the

number and pattern of isoenzymes of the parasites and the host

t i s sues . No previous report i s available on the isoenzymes of

G-3-PDH,

Glucose phosphate isomerase: (GPI)


Another enzyme of the g lyco lyt i c pathway i s glucose phos-

phate isomerase. This enzyme brings about internal rearrange-

ment of glucose-6-phosphate to give fructose-6-phosphate. On

histochemical l o ca l i zat ion i t i s seen that this enzyme i s absent

in the outer layer of the tegument o f G. crumenifer but moderate

94

a c t i v i t y i s observed in the suckers, caecae, testes , parenchy-

ma and musculature while inner layer of the tegument, ventral

pouch and lymph vesse ls show very l i t t l e enz3nne ac t i v i ty (Table

XVIII; Plate V, Figs. 1 -3 ) . In P. epiclitum only caecae give

intense reaction f o r GPI and inner tegumental layer possess

moderate act iv i ty which i s s l ight ly di f ferent from G, crumeni

f e r otherwise the rest of the regions show similar staining and

distr ibution of this enzyme. The overal l ac t iv i ty of GPI in

G. explanatum seems to be much lesser than the other two para-

s i t e s as evident by the intensity of staining observed in i t s

d i f f e rent regions. In the above case the ventral sucker i s com-

p le te ly negative; tes tes , v i t e l l a r i a , eggs and lymph vessels

show moderate ac t iv i ty while caecae, ovary and musculature have

l e s ser amount of th is enzyme.

No ear l i er histochemical study on the presence and d i s t r i -

bution of GPI in trematodes i s available. However, Yusufi and

Siddiqi (l978) studied the spec i f i c act iv i ty of GPI in G. cru-

menifer, S. in die a (P. epiclitiom) and Isoparorchis hypselobagri

(from Wallago attu) .


There are seven isoenzyme of GPI in G. crumenifer. Isoen-

zymes 1-4 are anodal whereas 5-7 migrate towards the cath^pde

(Plate XVI, Fig. A). The other parasite P. epiclitum shows the

presence of only f i v e isoenzymes of GPI. Bands 1 and 2 are near

the anode and the other three bands 3-5 are near the cathode

i o •p (0

o H cd «H 0) H o w

<D CO to

M M M

w PQ < E-,

0) -P CO P.

O •H •P W 0) s o CQ TJ O X! «H ft O

(U <0 o o ^ bO O § •H +» cd N •H O .3

(Q 

m W) bO W

-P •H >

I 0)

to -p

I C Cd o

E-i

CM >

(U CO o cO o

CO >

CO «

o

fH fn (U Q) ^ ^

CU 0) 3 CO O H

to 0) •P to CO (h CO PL,

+ +

+ +

+ + +

+

+ +

1

!x! u CO CU H <H >• •H G -P CU O g p -p to O CO O a

+ +

+ +

+ +

+ + +

+ +

+ +

+ +

+ +

+ +

95

+ + +

+ +

OJ H > O o o § h£l •H a

•J3 CO H

s CD

0) (0 +» o s; ti *

0) -p CO bO 0) S II

CU

CD CO CU X} o s

+ +

CU CO G OJ

+ + +

Plate XVI Electropherograras with corresponding densitometeric scans of polyacrylamide ge l s , stained f o r glucose phosphate isomerase ac t iv i ty . Numbers on the scans represent the bands. Arrows indicate the d irect ion of enzyme migration.

Figures:

A. Gastrothylax crumenlfer


C. Buffalo rumen


E. Buffalo l i ve r

97

(Plate XVI, Fig. B). The isoenzymes of GPI of the two amphi-

stome parasites o f buf falo rumen d i f f e r in number as well as

in re lat ive mobil ity. In rumen also the number o f GPI isoen-

zymes i s seven as observed in G, crumenifer (Plate XVI, Fig. C) .

Bands 1-5 are anodal whereas bands 5 and 7 move towards the

cathode. The posit ion of the isoenzyme bands of the two para-

s i t e s and their host tissue are also d i s t inc t ly d i f f e r e n t .

Interestingly the l i ve r parasite G. explanatum has only

four f ract ions of GPI (Plate XVI, Fig. D). Bands 1 and 2 are

anodal while bands 3 and 4 show cathodic a f f i n i t y . In the case

o f l i v e r there are seven isoenzymes of GPI. Isoenzymes 1-3

move towards the anode whereas 4-7 are cathodal bands (Plate

XVI, Fig. E). An interest ing observation in the case o f G.

explanatum and the host tissue l i v e r i s that bands 2-4 of the

parasite show a l i t t l e similarity with bands 3-5 of the host

t issue in their mobil ity. Instead of the above s imilarity the

niomber and pattern of the isoenzymes of the host t issues and

that o f the parasites d is t inct ly vary.

Agatsuma and Suzuki ( l98l ) reported that the number and

pattern of GPI isoenzymes d i f f ered not only between P. ohirai

and P. mivazakii but also between the parasite and the host(rat)

t i s s u e . But interest ingly , in yet another study on Fasciola sp.

no variations in the banding patterns between samples from ind i -

vidual f lukes were noted (Agatsuma,1981a). Further studies on

GPI isoenzymes on Paragonimus sp. , revealed considerable intra -

s p e c i f i c variations (Agatsuma,1981b,c). In yet another study

97

(Plate XVI, Fig. B). The isoenzymes of GPI of the two amphi-

stome parasites of buf falo rumen d i f f e r in number as well as

in re lat ive mobil ity. In rumen also the number of GPI i soen-

zymes i s seven as observed in G, crumenifer (Plate XVI, Fig. C) .

Bands 1-5 are anodal whereas bands 5 and 7 move towards the

cathode. The posit ion of the isoenzyme bands of the two para-

s i t e s and their host tissue are also d is t inc t ly d i f f e r e n t .

Interestingly the l i v e r parasite G. explanatum has only

four f ract ions of GPI (Plate XVI, Fig. D). Bands 1 and 2 are

anodal while bands 3 and 4 show cathodic a f f i n i t y . In the case

o f l i v e r there are seven isoenzymes of GPI. Isoenzymes 1-3

move towards the anode whereas 4-7 are cathodal bands (Plate

XVI, Fig. E). An interesting observation in the case of G.

explanatum and the host tissue l i v e r i s that bands 2-4 of the

parasite show a l i t t l e similarity with bands 3-5 o f the host

t issue in their mobil ity. Instead of the above s imilarity the

number and pattern of the isoenzymes of the host t issues and

that of the parasites d i s t inc t ly vary.

Agatsuma and Suzuki ( l98l ) reported that the number and

pattern of GPI isoenzymes d i f f ered not only between P. ohirai

and P. miyazakii but also between the parasite and the host(rat)

t i s s u e . But interest ingly , in yet another study on Fasciola sp.

no variations in the banding patterns between samples from i n d i -

vidual f lukes were noted (Agatsuma,1981a). Further studies on

GPI isoenzymes on Paragonimus sp. , revealed considerable intra -

s p e c i f i c variations (Agatsuma,l981b,c). In yet another study

98

de Boissezon and Jelnes (1982) observed mobility di f ferences

o f GPI isoenzymes of adult S. mansonl and S. rodhalni as well

as in their cercar ia l stage. These authors were of the view

that these charactersties might be useful f or a quick ident i -

f i c a t i o n of schistosome cercariae emerging from Biomphalaria.

Furthermore, Agatsuma and Suzuki (l983) showed that in the case

of S. mansonl and S. .japonicum there has been no di f ference in

the number and posit ion of the bands of the two sexes of the

same species. However, dist inct variations have been noted

between the two species as well as between the parasite and

the host (rat) t i ssue .

Aldolase*.


Aldolase i s also a key enzyme of g lyco lys is . It sp l i ts

the fructose 1,6-diphosphate molecule into dihydroxyacetone pho-

sphate and glyceraldehyde-3-phosphate. The histochemical resu-

l t s show that th is enzyme i s highly active in the suckers, ven-

t ra l pouch and testes of G. crumenifer whereas caecae and muscu-

lature have moderate act iv i ty (Table XIX; Plate V, Figs. 4 - 6 ) .

In the case of P. epiclitum the tegument shows s imilarity in the

intensity of staining and distr ibution, with the tegument of G.

crumenifer. There i s no major di f ference regarding the presence

and act iv i ty of aldolase in d i f ferent regions except the tegu-

ment (which i s negative) in G. explanatum and the other two am-

phistome parasites. Similarly no enzyme act iv i ty i s seen in the

99

v i t e l l a r i a , uterus and eggs of G, explanatum. However, high

aldolase act iv i ty has been found in the gonads while ventral

sucker and caecae give moderate reaction. In the past no a t -

tempt has been made to l o ca l i ze th i s enzyme by histochemical

methods in trematodes.


Aldolase ex is ts in the form six isoenzymes in G. crume-

n i f e r (Plate XVII, Fig. A). Bands 1-3 are anodal whereas the

other four bands 4-7 show cathodal migration. On the other

hand P. epiclitum also appears to have six isoenzymes of aldo-

l a s e . Here only band 1 i s anodal while the other f i v e bands

(2-6) move towards the cathode (Plate XVII, Fig. B). The ru-

men also possesses the same number of aldolase isoenzymes as

in the above two parasites. In th is case bands 1-3 move towa-

rds the anode and bands 4-6 show cathodal migration (Plate XVII,

Fig . C) . But no s imilarity except in the number has been ob -

served between the parasites and the host tissue isoenzymes.

Six isoenzymes of aldolase are also found in the case of

explanatum. Isoenzymes 1-3 are anodal in the ir migration

whereas bands 4-6 are near the cathode. (Plate XVII, Fig. D).

Though the number of isoenzymes of the three species i s iden-

t i c a l , there i s a c lear di f ference in their respective mobi l i -

t i e s . In l i v e r homogenate the number of isoenzymes i s seven.

Bands 1 and 2 move towards the anode, band 3 i s in the mid-gel

region while bands 4-7 are cathodal (Plate XVII, Fig.E).There i s

0) s o m o •H H X! Cd Pi § «H

B a •H o

•H X P H w M X Cd Q)

H a M o o

-Ci -a pq H < cd H o

o CQ CU G -P

O •H •H CQ -P Cd Cd u N cd

•H ft H CO o o

w w bO W

•P tD

-P •H >

I o; ::} fH o w cd

S H

I C Cd 0) B U a xi Du o

> T} a G 5 EH

(U cd o .

p a cd C •H H

1 u u 0) QJ p

0) cd O H

* * 1

+ 1

G 0) 0) to

+ + + +

o is; 11 ll

* + + +

(U > •r-l

•p + + + cd + + + t>0

+ 0) S

+ + + II + + + + + I

+ 0) + * * ^ +

II +

+ + + CU + + + -p

+ cd u <D

+ + + o + + s +

[l +

+ + + + + + + +

Q) 03 C CU 4-'

+ + + G + M +

II

§ 1 CU a u a 4-' H

cd OJ o •H > •p H tH p> H cd

CO > •H CO O O G CD •H •H O ro

p OJ P O H •H o a P CU P> P CO u D a R cd p Cd § CU u CO o OS cd cd •H CU o CU o

100

+ + +

PLATE V.

F i g . 1 . Gastrothylax cnAmenifer. Frontal section showing glucose phosphate isomerase a c t i -v i t y in the tegument (Teg), ora l sucker (OS"), parenchyma (P), testes (T) and i n -t e s t i n a l caeca ( i n t ) . X 400.

F ig ,2 . Paramphistomum epicl i tum. Frontal section showing glucose phosphate isomerase a c t i -v i t y in the tegument (Teg), in tes t ina l caeca ( Int ) and parenchyma (P) . X 400.

F ig ,3 . Gigantocotyle explanatum. Frontal section showing glucose phosphate isomer ase a c t i -v i t y in the tegvment (Teg), v i t e l l a r i a (Vit), parenchyma (P) and lymph vesse ls (Lv; , X 400,

F i g . 4 . Gastrothylax crumenifer, Frontal section showing aldolase ac t i v i ty in the ventral pouch (Vp) and oral suckea? (OS), X 400,

F ig ,5 . Paramphistomum epic l i tum. Frontal section showing aldolase a c t i v i t y in the ventral sucker (VS), tegument (Teg), lymph vessels (Lv) and in tes t ina l caeca ( i n t j . X 400.

Gigantocotvie explanatum. Frontal section showing aldolase a c t i v i t y in the tegument (Teg), o ra l sucker (OS), parenchyma (P) and lymph vesse l s (Lv), X 400

Plate XVII Electropherograms with corresponding densitometeric scans of polyacrylamide ge l s , stained f o r aldolase ac t iv i ty . Numbers on the scans represent the bands, Arrows indicate the direction of enzyme migration.

Figiires:

A. Gastrothylax crijmenifer


C. Buffalo rumen

D. Gigantocptyle explanatum


101

D

1 2

I

102

no resemblance in the number and posit ion of aldolase isoenzy-

mes of l i v e r and that of G, explanatum.

Electrophoretic d i f ferences between S. mansonl and S. r o -

dhaini have been studied by Fletcher et ( l 9 8 l ) , taking into

account the number and pattern of several enzymes including a ldo-

lase , of their cercar ia l forms. They reported that aldolase has

been one of the eight enzymes which showed identical number and

pattern of isoenzymes between the two species. No other report

on the isoenzymes of aldolase in relat ion to any other trematode

species i s avai lable .

Alcohol dehydrogenase; (A1 DH)


Alcohol dehydrogenase part ic ipates in a special ized termi-

nal process where i t acts on acetaldehyde to form ethanol. This

terminal process i f operative, i s a variant of the g lyco ly t i c

scheme.

Act ivity of A1 DH i s absent in the outer layer of the tegu-

ment of a l l the three species. Oral sucker, caecae and ventral

pouch of G. crumenifer; inner layer of the tegument, caecae and

tes tes of P. epiclitum and v i t e l l a r i a of G, explanatum show mode-

rate presence of th is enzyme whereas the other regions of a l l the

species under study have l i t t l e enzyme act iv i ty (Table XX; Plate

VI, Figs. 1 -3) .

Histochemical investigations on the presence of this enzy-

me in adult trematodes have not yet been made. There are some

103

reports on the presence of A1 DH act iv i ty in larval forms e . g . ,

Gercaria helvetica and cercaria of Sphaeridiotrema globulus

(see reference in Smyth and Halton,1983). This enzyme was also

reported from the sporocyst, miracidium, rediae and cercariae

of hepatica (Humiczewska,1975a,b,c,d).


In the present study A1 DH has been foxjnd in the form of

seven isoenzymes in G. crumenifer (Plate XVIII, Fig, A). Bands

1-3 are anodal, band 4 i s in the mid-gel region while bands 5 -

7 are cathodal. In P. epiclitiim, .only f i ve A1 DH isoenzymes

have been observed. Isoenzymes 1-3 show anodal migration whe-

reas bands h and 5 are almost in the middle of the gel (Plate

XVIII, Fig. B). However, in the case of rumen of buf falo there

are eight isoenzyme forms of A1 DH (Plate XVIII, Fig. C). Bands

1-3 move towards the anode while the other bands (4-8) have mo-

ved towards the cathode. The isoenzymes of the two parasites

and the host t issue (rumen) vary in the ir number as well as in

the pattern of migration.

In G. explanatum. as in G. crumenifer there are seven

A1 DH isoenzymes. Bands 1-5 show anodal migration while the

other two bands (6 & 7) move towards the cathode (Plate XVIII,

Fig . D). But the host tissue l i v e r possesses six isoenzymes,

which i s lesser than one from i t s parasite G. explanatum. I s o -

enzyme 1 i s nearest to the anode, band 2 i s in the middle of

the gel whereas bands 3-6 migrate towards the cathode (Plate

0) a o -p

CO •H Xi i-03 •H O

H ctJ QJ W «H

3

i-l pq <: Eh

CO c 0 to o O -H in W >1 CU a (U o XJ tj

H tH O o ^ o CO o cu H 4-> CO •H

to <H CO O CO

c P< o

P<

•H -P CO N •H H CO O o

m t>fl

-p

-M •H > I 0) 3 U o CO -P

I C CO 0) s fn >1 CO Pu o

CQ x) CO 5

> t>

•p c CD E s cu

> 0) CO o 0) CO o

CO * >

CO o

dJ OJ ^ b « CO •ri H

Js in 0) CU CO

O H

104

+ +

+ +

+ +

+ +

+ +

+ +

+ +

1 1 cu a

X a •p H CO cu o •H -p H VH -p H -p CO

CO >1 •H CO 0 0 G <v q •H •rH 0 TO

-P cu Pi 0 H •H o s P , CU -p P to f-. a a X CO •p u CO CO OJ in to o 5-1 CO CO CO •H CU o 0

c cu cu CO -p

o s

cu

-p

CO w CD S

II +

cu •p

CO

(U -a o

+ +

<u to c cu -p

+ + +

Plate XVIII Electropherograms with, corresponding densitometeric scans of polyacrylamide ge l s , stained f o r alcohol dehydrogenase a c t i v i t y . Numbers on the scans represent the bands. Arrows indicate the direct ion o f enzyme migration.

Figtires: A. Gastrothyiax crumenifer B. Paramphistomxjm epiclittim

C. Buffalo rumen

D." Gigantocotyle exolanatum E. Buffalo l i ve r

106

XVIII, Fig, E). Thus there i s a c lear dif ference in the num-

ber and mobility of isoenzymes of the parasite and the host

t i s sue . Similarly the number and posit ion of Al DH isoenzymes

d i f f e r among the three amphistome species. While the number

of bands i s similar in the case of G. crumenifer and G. expla-

natum« there i s variation in the posi t ion of the bands.

Nothing dehydrogenase; (NDH)


Nothing dehydrogenase i s not an enzyme i t s e l f . But 'NDH'

e f f e c t has been described as a curious phenomenon which usually

occurs when tetrazolium staining techniques are applied (Wil-

kinson, 1 970) . There are however suggestions that'NDH' e f f e c t

i s due to alcohol dehydrogenase (Ferguson,1980).

In the present investigation the sections o f the three

amphistome species (G. crumenifer. P. epiclitum and G. expla-

natum) were stained by using tetrazolium technique but without

any substrate. The results thus obtained are compared with

d i f f e r e n t i a l distribution of Al DH in the same species o f t r e -

matodes. Very l i t t l e ac t iv i ty has been observed in almost a l l

the regions of the three trematode species except ventral pou-

ch of G. crumenifer; caecae o f P. epiclitum and eggs of G. exp-

lanatum, where i t has moderate ac t iv i ty but the caecae of the

l i v e r parasite possess high avt ivity (Table XXI; Plate VI, Figs,

4 - 6 ) . No other previous report on NDH act iv i ty in trematodes

i s available

107

Electrophoretic observations have also been made on NDH

isoenzymes with a view to compare them with A1 DH isoenzyme

p r o f i l e .


In G. crumenifer, f i ve bands have been observed (Plate

XIX, Fig. a ) . Band 1 i s c learly anodal while bands 2-5 show

cathodal a f f i n i t y . Only three bands of NDH appear in the case

o f P. epiclitum. Al l the three bands show cathodal migration

(Plate XIX, Fig. B). Whereas the rumen shows the appearance o f

four NDH bands. Bands 1 and 2 are anodal while bands 3 and 4

migrate towards the cathode (Plate XIX, Fig. C) .

The other trematode parasite G. explanatum possesses s ix

NDH bands (Plate XIX, Fig. D). Band 1 i s anodal, band 2 i s

near the middle of the gel while bands 3-6 show cathodal migra-

t i o n , The l i v e r homogenate shows the presence of only four

bands of NDH. Band 1 i s anodal, band 2 i s in the mid-gel region

whereas bands 3 and 4 show cathodal a f f i n i t y (Plate XIX, Fig.E).

The number and pattern of NDH bands of G. explanatum and

that of buf fa lo l i v e r also d i f f e r considerably. Further, there

i s no resemblance between the isoenzyme bands of A1 DH and NDH,

obtained in the three amphiston© spec ies . However, three Al DH

and NDH bands (2-4) of the host t issue ( l i v e r ) and G. explanatm

have s l ight resemblance in their migration but certainly the NDH

bands show a very fa int appearence. I t i s quite possible that

the presence of alcohol (as a contaminant) in the tissue might

H X T) !>< >1

pq <1 EH

0 S O w •H

05 G O •H H CO 

Ki bO bO W

4J

-P •H >

I OJ O cn +3 3 cc S H I G CO (U S U CO ^ cu o

108

+ +

> + + + CO o •a CO fl

• + + + +

• (li + * * • +

>

(U CO c QJ i + + CO + + o +

C O + + + •

>

to + + + o

U U • P H - P H CO

CO & • H CO O O B <u X! G •H • H O CO Q ) P O H

• H O S P Q J • P P CO q X CO • P § O

CO O w ( 0 CO CO • H cu a Pu O

g <u TO

o

Q)

bO 0) is: II

0)

11 +

CD U xi o

+ +

0) CO g

+ + +

PLATE VI.

Fig .1 , Gastrothvlax crijmenifer. Frontal section showing alcohol dehydrogenase ac t iv i ty in the tegument (Teg), parenchyma (P) and testes (T) . X 400,

Fig .2 . Paramphistomum epiclitiJm* Frontal section showing alcohol dehydrogenase ac t iv i ty in the tegument (Teg), ventral sucker (VS), intest inal caeca (Int) and lymph vessels (Lv). X 400.

Fig .3 . Gigantocotyle explanatum. Frontal section showing alcohol dehydrogenase ac t iv i ty in the tegument (Teg), intest inal caeca ( Int ) , eggs (E) and lymph vessels (Lv), X 400,

Fig .4 . Gastrothvlax crumenifer. Frontal section showing nothing dehydrogenase ac t iv i ty in the tegument (Teg), ventral sucker (VS), ventral pouch (Vp) and parenchyma (P), X 400,

F ig ,5 , Paramphistomum epiclitijm. Frontal section showing nothing dehydrogenase ac t iv i ty in the tegument (Teg), parenchyma (P), in tes -t inal caeca ( Int ) , lymph vessels (Lv) and testes (T) , X 400,

Fig .6 , Gigantocotyle explanatum. Frontal section showing nothing dehydrogenase ac t iv i ty in the eggs (E) and intest inal caeca ( Int ) , X 400,

Plate XIX Blectropherograras with corresponding densitometeric scans of polyacrylamide g e l s , stained f o r 'nothing' dehydrogenase a c t i v i t y . Numbers on the scans represent the bands. Arrows indicate the direct ion o f enzyme migration

Figures:



C. Buffalo rumen


E. Biiffalo l i ve r

109

A - iU

110

be responsible f o r the appearence of NDH bands as suggested by-

Ferguson ( l980) . However, on the basis o f the present results

i t i s assumed that not only the presence o f alcohol but also

some other contributing factors may be the cause f o r the appear-

ence of 'NDH' e f f e c t .

Pho spho glucomutase: (PGM)

Hiatochemical study;

In the sequence of g lyco lyt i c pathway, PGM acts upon glu~

COse-1-phosphate to convert i t into glucose-6-phosphate which

can then be metabolised by either the g lyco ly t i c or pentose

phosphate pathway.

Histochemical examination of the parasite® f o r the prese-

nce and d i f f e r e n t i a l act iv i ty of PGM revealed that the outer

tegumental layer of a l l the three species has no enzyme a c t i -

v i t y . Highest enzyme act iv i ty , however, has been observed in

the oral sucker of G. crumenifer while the inner layer of the

tegument, ventral pouch, caecae and gonads show moderate prese-

nce of th is enzyme (Table XXII; Plate VII, Figs. 1 - 3 ) . In P.

epiclit\:un the enzyme act iv i ty i s l esser than G. crumenifer.

though the d i f f e r e n t i a l distribution i s almost the same in

both cases. But in G. explanatum the tegument (inner as well

as outer) i s completely devoid of PGM act iv i ty whereas the

other regions show somewhat similar l oca l i zat ion as in the

other two amphistomes. In G. explanatum the eggs have also

shown moderate PGM act iv i ty (Table XXII; Plate VII, Figs. 4 - 6 ) .

111

There i s no other report on the loca l i zat ion and d i f -

f e rent ia l distribution of PGM in trematode parasites . However,

the biochemical investigation on s p e c i f i c ac t iv i ty of PGM by

Yusufi and Siddiqi (l978) revealed uniformity in the enzyme

a c t i v i t y o f the two amphistomes v i z . , G. crumenifer and S. in -

die a (p. epiclitxjm).


Three PGM isoenzymes have been found in G. crumenifer.

A l l the three bands show cathodal migration (Plate XX, Fig, A).

In p. epiclitum the number of isoenzymes i s f i v e . Here again

a l l the bands move towards the cathode (Plate XX, Fig. B). The

host tissue rumen possesses s ix PGM isoenzymes and a l l the bands

show cathodal a f f i n i t y as was observed in the case of i t s two

parasites (Plate XX, Fig, C). Though the PGM isoenzymes of the

parasites as well as of the host t issue migrate towards the cath-

ode they are de f in i t e ly d i f ferent in their number and mobil ity.

explanatum has only four PGM isoenzymes which migrate

towards the cathode as has been the case with the other two pa-

ras i t es and the host tissue rumen (Plate XX, F ig . , D), The host

t issue l i v e r shows the presence of three isoenzymes of PGM and

again there i s no band in the anodal region. Band 1 i s in the

middle of the gel while bands 2 and 3 are towards the cathode,

(Plate XX, Fig. E). Though there i s a c lear di f ference in the

number and pattern of the isoenzymes of PGM among a l l the three

species of amphistomes as well as their host t issues but

H H

S M

6-t

0) a o -p •H Si p-s o

5 ^ o

E-i

a. >

0) cd o (V cd o

CO >

CO

o

c 0) a SI OJ

0) 0) fi !>.

u u CD 0) cd O H

112

+ +

+ +

+ +

+ +

+ + +

+ +

+ +

+ +

+ +

+ +

+ +

1 1 CD H !H a -P H cd 0) 0 •H > H tH 4J H to > •H to 0 0 CD •H •H 0 -P (1) P 0 •H 0 s p 0) -P to p a li § § u to 0 5-1 cd Cd Cd •H 0 04 C5

Cd

0)

a 0) d) to 4-' O S

cu -p cd bO 0) s II

(U IS

OJ cd in cu X )

+ +

0) to g -p 5

+ + +

Plate XX Electropherograras with corresponaing densitometeric scans of polyacrylamide gels , stained for phosphoglucomutase a c t i v i t y . Numbers on the scans represent the bands. Arrows indicate the direct ion o f enzyme migration.

Figures:



C. Buffalo rumen


E. Buffalo l i ve r

113

•i'

114

curiously , in almost a l l the cases, barring the l i v e r , the i s o -

enzymes move to the cathodal region.

The ear l i e r study on the PGM isoenzymes of the two s t ra i -

ns of S. intercalatum (one from lower Guinea and the other from

Zaire) by Wright et a l . ( 1979) revealed marked d i f ferences be t -

ween the two. Furthermore, PGM has been reported to have two

isoenzyme forms in F. hepatica (Agatsuma and Suzuki,1981). In

yet another study Fletcher £t ( l 98 l ) found three forms of

PGM in Schistosoma sp . , and suggested that this enzyme i s the

best f o r diagnostic d i f f erent ia t ion . However, they could not

f ind any di f ferences in the mobi l i t ies of the cercar ia l and

adult isoenzymes, de Boissezon and Jelnes (l982) studied the

variat ions between the iso lates of cercariae and adults of S.

mansonl and S. rodhaini and suggested that the PGM isoenzyme

p r o f i l e might be useful f o r a quick ident i f i cat ion of sch is to -

some cercariae emerging from Biomphalaria. Furthermore, Agat-

suma and Suzuki (l983) demonstrated di f ferences in the nimber

and posit ion of PGM isoenzymes of S, .japonlcum and S. man son i

as well as d i f ferences with the host (mouse) blood and muscles.

Hexokinase; (HK)


Hexokinase i s the most widely distributed enzyme which

normally catalyses the phosphorylation not only o f D-glucose

but also of many other hexoses and hexose derivatives, inc lu -

ding D-fructose, D-mannose and D-glucosamine.

115

The histochemical results , presented here, revealed that

the tegument of the three species except the inner tegumental

layer of P. eplclitum i s negative f o r HK ac t iv i ty . Lymph ves-

se l s of G. crumenifer stained intensely while the other regions

show moderate to weak enzyme ac t iv i ty . Organs other than the

tegument, where no enzyme act iv i ty i s observed are ventral pou-

ch and v i t e l l a r i a of G. crumenifer; musculature and v i t e l l a r i a

of_P. epiclitum and caecae, v i t e l l a r i a and uterus of G. expla-

natum. However, the eggs of the l i v e r parasite possess high

enzyme act iv i ty (Table XXIII; Plate VII, Figs. 4 - 6 ) . The pre-

sent findings are in agreement with those reported by Sharma

and Mandawat (l983) on HK distribution in G. tigrinum.


There are three isoenzyme forms of HK in G. crumenifer

(Plate XXI, Fig. A). Band 1 and 2 migrate towards the anode

while band 3 shows cathodal migration.Three bands of ac t iv i ty

also appear in the case of P. epiclitum. Band 1 i s near the

middle of the gel whereas bands 2 & 3 are cathodal (Plate XXI,

Fig. B). The rumen also shows the presence of only three i s o -

enzymes in the form of three bands, a l l of which move towards

cathode (Plate XXI, Fig, C) , Though the isoenzyme bands of

the two parasites and their host tissue are identical in num-

ber, they d is t inc t ly d i f f e r in their mobi l i t ies .

In the case of G. explanatum there appear ten isoenzyme

bands of HK. First four bands ( l - 4 ) are near the anode band

H

0) e o -p •d o ^ H p. CO a VI cd «H

o 0) -rH w a

3 < Eh tH O

a o •H -P

-P (0 QJ s O xJ

w QJ -H •H n nj CO CO isi p, •H H CO o o J

152

w bO bO W

•H > I o; ^ b O CQ +3 •S CO S H I C CO 0) S COX! o

• > CO O T) O

CO C cS • EH

• Oi •

> CU CO o CU CO o

• CO •

> • CO •

o

u u d) 0 c ^ C CO C •H H Q) s t. JH P > 0) 3 CO EH O H

+ +

+ +

+ +

+ +

+ +

+ +

+ +

+ +

+ + +

+ +

X IH s •P CO o •rH H <H +5 H to > •H CO o CU .-c! •H •H -p CU P •H p a P •

O O O § uq •H o

+5 CO H s Q)

C CU 03

O IS

(U

CO fc>£) <U II

It +

0 -P CO U Q) X}

+ +

0) CO fl (U -p

+ + +

PLATE VII .

Fig.1» Gastrothylax criMenifer. Frontal section showing phosphoglucomutase ac t iv i ty in the tegument (Teg), oral sucker (OS), ven-tra l pouch (Vp) and parenchyma (P). X 400,

F ig .2 . Paramphistomum epiclitum. Frontal section showing phosphoglucomutase ac t iv i ty in the tegument (Teg), parenchyma (P) and testes (T). X 400.

F ig ,3 . Gigantocotyle explanatum. Frontal section showing phosphoglucomutase ac t iv i ty in the eggs (E), v i t e l l a r i a (Vit) and testes (T), X 400.

F ig .4 . Gastrothylax crumenifer. Frontal section showing hexokinase act iv i ty in the tegu-ment (Teg), ventral pouch (Vp), testes (T), and parenchyma (P). X 400.

F ig .5 . Paramphistomum e p i c l i t i ^ . Frontal section showing hexokinase act iv i ty in the tegu-ment (Teg), intest inal caeca (Int) and lymph vessels (Lv). X 400.

F ig .6 . Gigantocotvle explanatum^ Frontal section showing hexokinase act iv i ty in the eggs (E), testes (T) and parenchyma (P). X 400.

m C f ' M

• •T-'v/''':'- I . " . ^^

Plate XXI Electropherograms with corresponding densitometeric scans of polyacrylamide ge ls , stained for hexokinase ac t i v i ty . Numbers on the scans represent the bands. Arrows indicate the direct ion of enzyme migration

Figures:


B. Paramphlstomum epiclitum

C. Buffalo rumen


E. Buffalo l i ver

117

s

A J \ i i

118

5 i s in the middle region o£ the gel while bands 6-10 move t o -

wards the cathode (Plate XXI, Fig. D). But the host tissue

possesses only four isoenzymes (Plate XXI, Fig. E) . Band 1 i s

anodal, band 2 i s in the middle of the gel whereas band 3 and

4 show cathodal migration. The number and posit ion of HK i s o -

enzymes of the parasite and the host tissue are again found

to have no s imi lar i ty .

Fletcher e^ a l . ( l98 l ) found no variation in the number

and pos i t ion of HK isoenzymes of S, mansoni and S. rodhaini.

This enzyme has also been found to occur in two isoenzyme

forms in the cercariae and adults of S. mansoni and S. rodh-

a in i (de Boissezon and Jelnes,1982). Furthermore, Agatsuma

and Suzuki (l983) reported the presence o f three isoenzymes of

HK in S. .iaponicum and S, mansoni. They, however, observed

that the relat ive mobi l i t ies of the isoenzymes in the above two

species were d i s t i n c t l y d i f f e rent . Thus i t i s c lear that not

only c lose ly related species show a d i s t inc t ly d i f f e rent number

and pattern of isoenzymes but even a single species from d i f -

ferent parts o f the world, has been reported to have unidenti-

ca l isoenzyme p r o f i l e .

119

SDS-PAGE of general proteins;

The results o f the SDS-polyacrylamide disc gel e l e c t r o -

phoresis of the general proteins revealed fourteen bands in the

case o f G, crumenifer. Bands 1-8 are anodal, band 9 i s in the

middle of the gel while bands 10-14 show cathodal migration

(Plate XXII, Fig, A) . In P. eplclitum the number of bands i s

twelve. Bands 1-6 move towards the anode, bands 7 and 8 are in

the mid-gel region whereas bands 9-12 have moved in the catho-

dal direct ion (Plate XXII, Fig. B). The rumen shows the pre -

sence o f sixteen protein f rac t i ons . Bands 1-9 are anodal while

bands 10 and 11 are in the middle of the gel and 12-16 show

cathodal migration (Plate XXII, Fig, C) ,

In the case of l i v e r parasite G, explanatum, the tota l

number of bands i s eighteen. Bands 1-7 show anodal migration,

8 and 9 are in the mid-gel region while the remaining nine bands

(10-18) moved towards the cathode (Plate XXII, Fig, D), The

l i v e r has been found to possess twentyone protein bands, the

f i r s t ten bands ( l - lO) migrate towards the anode, bands 11 and

12 are in the middle of the gel while the other bands (13-21)

have moved in the cathodal direct ion (Plate XXII, Fig. E),

The number and the re lat ive mobi l i t ies of protein bands

of the rumen parasites (G, crumenifer and P, epiclitum) are

d i f f e r e n t . The rumen tissue also revealed d i s t inc t ly d i f ferent

number and relative mobi l i t ies of protein bands as compared with

Plate XXII Electropherograms with corresponding densitometeric scans of polyacrylaraide ge l s , stained f o r general proteins. Numbers on the scans represent the bands, Arrows indicate the direct ion of protein migration.

Figures; A. Gastrothvlax cruraenifer B. Paramphistomum epiclitum

c . Buffalo rumen D. Gi^antocotyle explanatum

E . Buffalo l i ve r

120

a H iz «

121

i t s parasites . Similarly, d i f ferences have been obtained in

the case of G, explanaturn and i t s host tissue ( l i ver ) protein

p r o f i l e . Thus i t could be said that each parasite as well as

the host tissues possess a s p e c i f i c number of protein bands

which show a particular pattern of migration.

The whole worm homogenates have ear l ier been studied by

several workers to obtain the d i f f e r e n t i a l protein pattern of

the d i f f e rent species through electrophoresis and i s o e l e c t r i c

focuss ing . Yoshimura et (l970) obtained 2l bands, 19 of

them reproducible from the whole worm homogenate of P. k e l l i -

c o t t i . The densitometeric examination gave 8 characterstic

peaks. The protein pattern of P. k e l l l c o t t i was found to be

d i f f e r e n t from P. westermani, P. ohira i , P. iloktsuenensis and

P. miyazakii. The two morphologically similar species v i z . P.

k e l l i c o t t i and P. miyazakii. showed similar patterns near both

ends of the gel but the mid-gel area showed dist inct d i f f e r e n -

c e s . Dissimilarit ies in the protein spectrum of some amphisto-

mes namely C. calicophorum. L. scotiae and G. crumenifer have

been noted by Klimenko and Velichko ( l972) . The v a l i d i t y of

the three species of trematodes v i z . P. microbothrlum, P. cervi

and P. microbothreoides was confirmed by Osikovosky et a l .

( l978) on the basis of the patterns of water soluble protein

extracts of these species. Both one- and two dimensional e l e c -

trophoret ic separation of S. mansoni proteins Atkinson and

Atkinson (l982) reported that homogametic males produce f ive

polypeptides while heterogametic females did not show the same

122

pattern in protein p r o f i l e .

The present investigation i s in agreement with the f i n d -

ings of ear l i er workers part icularly that of Yoshimura et a l .

( l970) and Klimenko and Velichko (l972) regarding the s p e c i f i c

variat ions in the protein p r o f i l e of d i f ferent species obtained

by using polyacrylamide gel e lectrophoresis . The present study

on the amphistomes belonging to three d i f ferent genera and i n -

habiting two d i f f erent habitats in the same host revealed mar-

ked d i f ferences in the general protein pattern. Therefore, i t

can be concluded that protein p r o f i l e alone, can be used in dis-

tinguishing the d i f f erent amphistomes at generic l e v e l .

S U M M A R Y A N D C O N C L U S I O N S

123

The present histocliemical and electrophoretic invest iga-

t ions have been made on a comparative basis . The amphistome

parasites selected f o r th is study belonged to three distinctly-

d i f f e rent genera but parasit izing the same host Bubalus bubalis

(domestic buf fa lo ) and inhabiting two d i f ferent regions. The

parasites were Gastrothylax crumenifer; Paraiaphistomum e p i -

clitum from the rumen and Gigantocotyle explanatum from the

l i v e r . This study i s divided into three parts v i z . , general

histochemistry by paraf f in technique, histoenzymology by cryo-

tomy and isoenzyme studies by polyacrylamide gel electrophoresis .

In general histochemistry, acid mucopolysaccharides, amyloids,

co l lagen, e last in , f i b r i n , tryptophan and nucleic acids (DNA

and RNA) have been l oca l i zed . The histoenzymological and e l e c -

trophoretic studies include l4 important enzymes (8 dehydroge-

nases) namely glucose-6-phosphate, 6-phosphogluconate, malate,

succinate, c<^glycerophosphate, hexose-6-, glyceraldehyde-3-phos-

phate, alcohol and nothing dehydrogenases and fructose l , 6 - d i -

phosphatase, glucose phosphate isomerase, aldolase, phosphoglu-

comutase and hexokinase.

The main purpose of the histochemical study was not only

to see the presence or absence of various biochemical components

including enzymes but also to observe the d i f f e r e n t i a l d is tr ibu-

t ion of these substances in d i f ferent organ systems and to sug-

gest their possible ro le in the metabolism of the parasites

124

under study. This could be achieved af ter thorough microsco-

p i c a l examination of the stained sections in f o r d i f -

f e rent ia l a c t i v i t i e s of the above mentioned enzymes. In ad-

di t ion to this the same enzymes were also studied by polyacry-

lamide gel electrophoresis (PAGE) to observe their isoenzyme

p r o f i l e in the parasites and their respective host t i ssues .

The aim of this study of isoenzyme p r o f i l e of various enzymes

was however not to investigate the gene frequency of a part icu-

lar enzyme but i t was done with a view to f ind out as to how

the isoenzymes help the trematode to regulate their metabolism

in d i f ferent ecophysiological conditions and also the possible

use of the p r o f i l e ( s ) in the biochemical taxonomy. Further,

th i s study was also undertaken in order to see the e f f e c t of

the habitat on the parasite l iv ing in d i f ferent habitats in a

single host, and also to establish the biochemical basis of the

host-parasite relat ionship as well as the degree of dependence

of the parasite on the host.

The results of general histochemistry show that acid muco-

polysaccharides are present in higher amounts in the outer layer

o f the tegument of G. crumenifer and P. epiclitum than G. expla-

natum. The testes and uterus of the l i v e r parasite also show

intense staining f o r the presence of acid mucopolysaccharides.

I t i s suggested that the high acid mucopolysaccharide accumula-

t ion in the tegument of the two rumen parasites i s to protect

them against their continuous exposure to certain digestive en-

zymes and continuously changing environment of rumen due to the

125

promiscuous feeding of the host. Since acid mucopolysaccharides

are also known to help in osmoregulation, therefore, i t i s sug-

gested to be a biochemical adaptation on the part of the para-

s i t e s . The other araphistome, G. explanatum inhabits the b i l e

ducts where i t i s continuously exposed to the b i l e secretions

(containing b i l e sa l t s ) which are known to possess detergent

e f f e c t , consequently resulting in the depletion of this subs-

tance from the tegument.

The d i f f e r e n t i a l distribution of amyloids in the three

trematodes i s almost similar except that the tegument of the

rumen parasites possess moderate amounts whereas the tegument

o f the l iver parasite i s negative. Amyloids are also known to

be resistant against proteo lyt ic a c t i v i ty (Pearse,1968)> and

hence i t seems relevant that only the worms inhabiting the ru-

men and threatened by the proteo lyt i c enzymes, possess higher

amounts of amyloids than the l i ver parasite which faces least

p o s s i b i l i t y of being exposed to such enzymes. The histochemi-

ca l l o ca l i zat ion of structural proteins v i z . , col lagen, e last in

and f i b r in did not show any s igni f i cant variation in their d i s -

tr ibut ion in the body organization of the three species . Though

there i s no conclusive evidence to suggest there possible fun-

c t i ona l role in the trematode organization, i t i s l i k e l y that

col lagen f i b res may provide mechanical strength to the tissue

of the parasite in order to maintain the integri ty of the c e l l s .

I t i s also possible that the col lagens, i f they e x i s t , having

126

d i f f e rent chemical configuration, may help the parasite in the

egg-she l l formation, immunogenic responses and in providing

strngth to the muscles. Elastin seems to maintain the tone

of muscles and e l a s t i c i t y to recover the original shape a f ter

the wave of contraction and relaxation. Fibrin might also be

functioning in association with collagen and e las t in . An im-

portant protein bound amino acid tryptophan has been found in

the d i f f e rent regions o f a l l the three species. I t i s d i f f i -

cu l t to assign any s p e c i f i c function to tryptophan except that

i t may be involved in the s tab i l i zat ion of egg-shell proteins

or may be a integral component of other long chain proteins.

The most important biochemical components of the tremato-

des are the nucleic acids (DNA and RNA), The l o ca l i za t i on of

nuc le ic acids i s almost the same in the three species under

study. The presence o f DNA and RNA in the caecae and i t s con-

tents indicate that they might be derived from the host in as -

soc iat ion with other substances during feeding. Further the

ubiquitous presence of nucleic acids in d i f ferent structures,

part i cular ly the gonads, suggest that possibly these are p lay-

ing an important role in growth, reproduction and in protein

synthesis.

Histochemical l oca l i zat ion and d i f f e rent ia l distr ibution

o f certain enzymes in the body organization of the three amphi-

stome species i s more or less similar and has indicated that

g l y c o l y s i s , pentose-phosphate shunt and TCA cycle may be opera-

t i ve in the parasites under study. But J^st on the basis of

127

mere presence or absence of an enzyme i t can not be conc lus i -

ve ly said whether the cycle i s actually operative or not . How-

ever, the occurrence of d i f ferent enzymes in the tegiament of

the parasites provide an idea of i t s active role at the host -

parasite interphase and thus support the hypothesis that the

tegument of trematodes i s metabolically act ive. The pos i t ive

reaction in the inner l ining of the ventral pouch of.. G. cru-

menifer also shows i t s partic ipation in the metabolic ac t iv i ty .

Only HK and PGM were however absent in the tegument of G. expla-

natum.

The electrophoretic studies have revealed that these enzy-

mes occur in the form of varying nxjmber of f ract ions ( isoenzy-

mes) in the parasites as well as in their host t i ssues . - The

bands showing the presence of isoenzymes not only d i f f e r in num-

ber f o r each of the three species of amphistomes and.their c o r -

responding host t i ssues but also d i f f e r considerably in their

r e la t ive mobi l i t ies . The number of isoenzymes of d i f f erent en-

zymes of G, crumienifer, P. e-piclit\jm. G. explanatum and the host

t i s sues (rumen and l i v e r ) have been summarized. The relat ive

mobi l i t ies of isoenzymes of SDH, H-6-DH, gly-3-PDH, PGM and GPI

can be used as a va l id t oo l in the f i e l d of biochemical taxono-

my of amphistomes.

Close resemblance which has been noticed in the number of

isoenzymes of certain enzymes of the host tissue and their c o -

rresponding parasite species leads to the conclusion that there

i s some biochemical basis of host-parasite relationship. Whereas

128

from the diss imilar ity in the number of isoenzymes of certain

other enzymes, i t could be inferred that these parasites might

have some of their own independent enzymes and/or enzyme system

( s ) . Such variations may also be the result of some biochemical

adaptation on the part of the v/orms due to their paras i t i c mode

o f l i f e . As their larval stages pass through certain intermedi-

ate hos t ( s ) , some of their enzymes may be retained by their

adult forms even af ter reaching the def in i t ive host. Moreover,

i t i s also possible that such enzymes of the larval stages which

might have now become inactive due to the change in the eco -

physio logical conditions in the two di f ferent habitats as well

as change in the nutrit ional requirements of the worms, are

s t i l l capable of appearing on the ge ls .

Another thing which becomes c lear i s that the riJinen and

l i v e r of the same host possess d i f f erent number o f isoenzymes

o f most enzymes under study. This i s perhaps due to their d i f -

ferent physico-chemical nature and according to s p e c i f i c funct -

ions they perform. In some cases l ike SDH and GPI, the two host

t i s sues have however,been found to possess equal number of i s o -

enzymes in both cases. Whatever may be the case, i t i s quite

l i k e l y that the parasites being dependent on the host for their

nutr i t ion may also be taking pr iv i lege of the host enzymes.

Thus similar mobi l i t ies of some of the isoenzymes of the host

t i s sues and their respective parasites have indicated the i n f l u -

ence of the habitats on the worms and this shows the intr icate

nature of their host-parasite relationship.

129

The three amphistome species showed spec i f i c isoenzyme

p r o f i l e f or each enzyme but more marked di f ferences have been

noted in the number and relat ive mobi l i t ies of SDH, Fr1,6'-DP,

PGM and GPI isoenzymes. Hence these enzymes are presumed to

help the workers in the f i e l d of biochemical taxonomy, p a r t i -

cu lar ly in the taxonomic characterization of amphistomes which

require immediate attention because there are certain contro-

vers ies regarding the va l id i ty of several genera and species.

Furthermore, since the protein composition of an organism r e f -

l e c t s i t s genetic const i tut ion, separation of general proteins

by electrophoresis may also prove to be a valuable taxonomic

t o o l in studies of a variety of parasites . However, further

research i s required to be carried out on isoenzyme as well as

on general protein p r o f i l e s of a number of d i f f erent species

o f amphistomes in d i f f e rent populations of the same and d i f f e r -

ent host origin and also from d i f f e rent geographical regions

with more sophisticated techniques l ike i s o e l e c t r i c focussing

in order to evolve some standard enzyme markers f o r the b i o -

chemical taxonomy of the amphistome parasites.

130

APPENDIX A

Incubation media:

1. Glucose-6-phosphate dehydrogenase (G-6PDH)(E.C.1.1,1.49) (Shaw and Prasad,1970):

NADP 3 0 . 0 0 mg Nitro BT 2 0 . 0 0 mg P M S 0 2 o 0 0 m g

0 . 5 M tris-HCl pH 7.1 2 5 o 0 0 ml N ag Glue o se-6-pho sphate-H g O 2 0 0 o 0 0 m g

H O 90,00 ml

Gels were incubated at 37°C f o r 2V2 h, washed with d i s t i l l e d water and f ixed and stored in 7% acetic acid.

2. Malate dehydrogenase (MDH) (E .G .1 . l . l . 37 ) (Shaw and Koen, 1964):

Substrate: 1M Na L-malate

L-malic acid 13.40 g 2M NagCO H O (248g / l i t . ) 00.49 ml

Stain:

H2O to 100.00 ml

NAD 5O0OO mg NBT 30.00 mg PMS 02.00 mg .1M Na L-malate pH-7.0 10.00 ml .5M tris-HCl pH-7o1 15.00 ml H O 70 .00 ml .iMNaCN 05.00ml

131

Gels were incubated f o r 4 h at 37°C, washed and f ixed

with 7% acetic acid.

3. Succinate dehydrogenase (SDH, E.G.1.3.99.1; Pearse,1968):

Stock buffered succinate; Combine equal volumes of 0,2M phosphate buf fer

(pH-7.6) and 0,2M sodium succinate.

Incubating medium;

Stock succinate solution 10,00 ml Aqueous NBT (img/ml) 10.00 ml

Gels were incubated f o r 1 h at 37°C, washed and f i xed .

4 . Alcohol dehydrogenase (AIDH, E .C . l . 1 . 1 .1 ; Shaw and Koen, 1965):

NAD 50.00 mg NBT 30.00 mg PMS 02.00 mg .iMNaCN 05.00ml Ethanol (95%) 03.00 ml .5M tris-HCl pH-7.1 15.00 ml HgO 77.00 ml

Gels were incubated at 37°C f o r 3 V 2 h, washed and and f ixed in 1% acet ic acid.

5. 5-phosphogluconate dehydrogenase (5-PDH, E .G.1 . l .1 .44 ; Shaw and Prasad,1970):

Incubation medium;

NADP 20.00 mg NBT 25.00 mg PMS 02.00 mg Na^6-phosphogluconate 200.00 mg

132

.5M tris-HCl pH-7.1 10.00 ml H O 86.00 ml

Gels were incubated at 37°C for 2V2 h, washed and

f i xed in 1% acetic acid.

6 . Hexose-6-dehydrogenase (H-6-DH, E . G . l . l , l . 4 7 ; Shaw and Koen,1968a,b)s

Incubation medium;

NADP 50.00 mg NBT 50.00 mg PMS 02.00 mg Tris buf fer pH-6.8 10.00 ml 1M Galactose phosphate 05.00 ml H O 85.00 ml

Gels were incubated at 37°C f o r 3 h, washed and f ixed in 1% acetic acid.

7. glycerophosphate dehydrogenase (K-GPDHjE.C.1.1.1.8; Shaw and Prasad,1970):

Substrate; 1M Na^K^-glycerophosphate, pH-7.0 21.6 g H2O 100.00 ml

Dissolve NaK-glycerophosphate in 50 ml of water, adjust pH-7.0 with 1N HCl, make up to 100.00 ml.

Incubation medium; NAD 50.00 mg NBT 30.00 mg PMS 02.00 mg 1M Na-^glycerophos-phate pH-7.0 10.00 ml H2O 70.00 ml .iMNaCN 05.00ml

133

.5M tris-HCl pH-7.1 15.00 ml

Gels were incubated f o r 2} 12. h at 37°C, washed and

f i xed in 7% acetic acid.

8. Glyceraldehyde-3-phosphate dehydrogenase (G-3-PDH,E.C.1.2. 1.12; modification of the method of Williams,1954):

Substrate; Naofructose1,5-dipbosphate-6H|0 545.00 mg Aldolase" 00.60 ml (lOO Units) .5M tris-HCl pH-7.1 04.00 ml H O 06.00 ml

Mix and incubate substrate mixture at 37°C f o r 30 min.

Incubation medium;

NAD 50.00 mg NBT 50.00 mg PMS 02.00 mg Na2HA 0 150.00 mg Substrate 10.00 ml .5M tris-HCl pH-7.1 20.00 ml H O 70.00 ml

Gels were incubated at 37°C f o r 5 h, washed and f ixed

in 7% acetic acid.

9. Nothing dehydrogenase (NDH) Incubation medium;

NAD 30.00 mg NBT 20.00 mg PMS 02.00 mg .5M tris-HCl pH-7.1 15.00 ml H O 90.00 ml .iMNaCN 05.00ml

134

Gels were incubated in the above mediiim without any

substrate f o r 3 h at 37°C, washed and f ixed in 7% acet i c acid.

10, Glucose phosphate isomerase (GPI, E.G.5.3 .1 .9 ; Delorenzo

and Ruddle,1969):

Incubation medium;

NADP 10.00 mg NBT 10 .00 mg PMS 01.00 mg MgCl2 .1M tris-HCl pH-8.0 100.00 ml G-6-PDH 05.00 jal Fructose-6-phosphate 160.00 mg

Gels were incubated f o r 2 h at 37°C, washed and f ixed in 1% acet ic acid.

11. Fructose1,6-diphosphatase (Fl ,6-DP,E.C.3.1.3.11; Stout, 1969):

Incubation mediumt

NADP 15.00 mg NBT 20.00 mg PMS 02.00 mg .5M tris-HCl pH-7.5 lO.OO ml MgS0^7H20 250.00 mg 2-mercaptoethanol 0.002 ml Fructosei,6-phosphate 20.00 mg H O to 100.00 ml

Add just before use: 40 units of phosphohexose i s o -merase and 40 units of glucose-6-phosphate dehydrogenase.

Gels were incubated f o r 3 h at 37°C, washed and f ixed in 7% acetic ac id .

135

12. Phosphoglucomutase (PGM, E.G.2 .7 .5 .1 ; Spencer et a l . , 1964 ) :

Incubation medium;

NAD? 10.00 mg NBT 20.00 mg PMS 01.00 mg MgCl SH^O 200.00 mg Na^glwcose1-phosphate-H O 600.00 mg

G-6-PDH 0.035 ml (80 units) .5M tris-HCl pH-7.1 10.00 ml H O 90.00 ml


in 7% acet ic acid,

13. Hexokinase (HK, E.G.2 .7 .1 .1 ; Eaton ^ .1966):

Incubation mediijmt

NADP 25.00 mg NBT 20.00 mg PMS 03.00 mg Glucose 9O0OO mg ATP 25.00 rag MgCl26H20 21.00 mg G-6-PDH 0.035 ml (80 units) .5M tris-HCl pH-7.1 10.00 ml H O 90.00 ml


in 7% acet ic acid.

14. Aldolase (E.G.4o1.2.13; Shaw and Prasad, '1970):

Incubation medium;

Na2fructose1,6-diphosphate 5H2O 545.00 mg

136

G-3-PDH 0.60 ml (lOO units) NAD 50.00 mg NBT 30.00 mg PMS 02.00 mg .5M tris-HCl pH-7.1 10.00 ml Na2As0^.7H20 I50o00 mg H O 90.00 ml


in 1% acet ic ac id .

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P U B L I C A T I O N S

HELMINTHOLOGIA, 21: 21—31, 1984

Histochemical and histoenzymological studies on the metacercariae of Clinostomum complanatum (Trematoda: Digenea)

S. MAQBOOL ALAM, WAJIH A. NIZAMl

Section of Parasitology, Department of Zoology, Aligarh Muslim University, Aligarh 202001, India

Received November 6, 1982

Summary

Histochemical and histoenzymological studies were carried out on the metacercariae of Clinostomum complanatum, from the body cavity of the fresh water fish, Trichogaster fasciatus, in relation to the following substan-ces : Glycogen (Best's carmine and PAS), acid mucopolysaccharides (alcian blue), amyloids (Congo red), proteins (mercury-bromophenol blue), colla-gen (Mallory's aniline blue), myelin and fibrin (Gomori's trichrome stain and Van Gieson's picrofuchsin), lipids (Sudan Black B), DNA (Feulgen and methyl green pyronin), DNA and RNA (methyl green pyronin).

Acid phosphatase, alkaline phosphatase, lipase and non-specific esterases were localised in frozen sections. Differential distribution and possible functional role of these biochemical components in the metabolism of the metacercariae were also discussed.

Key words: histochemistry; histoenzymology; Clinostomum meta-cercaria

Introduction

Histochemical observations on the distribution of biochemical components and enzymes in trematodes have been restricted mainly to the adults and very fragmentary information is available on their larval stages.

The progenetic metacercaria of Clinostomum complanatum possesses well defined organ systems, including the reproductive organs but without any eggs. However, the metacercaria shows high motility and active metabolism (Thomas and Gallicchio, 1967; Siddiqui and Nizami, 1981), which can easily be compared with other adult trematodes.

The present investigation on the metacercaria is an attempt to localize the various biochemical constituents and some enzymes, in order to provide possible clues to their biological significance in the transformation from metacercaria to adult.

21

Material and methods

Metacercariae of Clinostomum complanatum were collected from the body cavity of the fish, Trichogaster fasciatus. They were washed several times with physiological saline in order to remove debris, fixed in different fixatives and specific histochemical techniques were applied. Fixation, embedding, sectioning and staining methods employed are given in Table I. Some metacercariae were quickly mounted on a chuck of a freezing microtome by using Tissue-Tek II compound (Lab-Tek Product, U.S.A.), for embedding of frozen material.

Results and discussion

Histochemical studies reveal that glycogen is deposited mainly in the parenchyma, musculature, suckers and less so in various other regions (Table 2). These findings are in agreement with the earlier reports on glycogen distribution and its metabolic significance in adult trematodes (Erasmus, 1972; von Brand, 1973).

The functional role of glycogen is well established in helminth parasites. Being an energy source, glycogen would naturally be associated with the sites of metabolic or muscular activity. Furthermore the presence of glycogen in the parenchyma surrounding the gonads (Plate I)

Table 1

Histochemical and hisloenzymoiogical techniques employed

Tissue constituents

Fixation Embedding Section

thickness

Polysaccharides: Glycogen Camoy's / AFA Bouin's Paraffin wax 7 nm Acid Mucopolysaccharides do do do Amyloids do do do

Proteins: General 10 % buffered formalin do do Collagen Carnoy's / AFA do do Myelin and fibrin do do do

Lipids :

General 10% buffered do do Nucleic acids:

DNA and RNA do do do Enzymes:

Alkaline phosphatase Cold (4°C) acetone Frozen gelatin 12 nm Alkaline phosphatase (Tissue-Tek)

Acid phosphatase 10% buffered (cold) . formalin

do do

Non-specific do do do esterases Lipase do do do

22

Table 1 (Continuation)

Slaining techniques Control References

Periodic acid Schiff (PAS) Diastast Grimstone and Skaer (1972)

Alcian blue None Drury and Walmgton (1967) Congo red None Drury and Walmgton (1967)

Mercury-bromophenol blue None Pearse (1961) Mallory's aniline blue None McManus and Mowry (1960) Gomory's rapid one step trichrome None Pearse (1961) stain/Van Gieson's picrofuchsm

Propylene glycol-Sudan method Methanol Pearse (1961) chloroform 60 °C

Methyle green pyronin/Feulgen None Pearse (1961)

a-glycerophosphate ' 90°Cfor 10 mm Pearse (1961) H)%NaF

Lead nitrate do Pearse (1961) S-bromoindoxil acetate 90°C Pearse (1961) Tween 80 90°q Pearse (1961)

shows Its importance as an energy reserve, for gonads to maintain growth and to reach functional stage Glycogen has berti described as a basic substrate for growth and reproduc-tion in cestodes (Read and Simmons, 1963) Due to the absence of a well defined circulatory system, the dependence of gonads and the surrounding parenchyma on glycogen is essential and obvious (Halton, 1967a) Furthermore, Nacheva (1975a) histochemically showed a glycogen is utilized in the concentration of muscles, confirming the utility of glycogen in the metabplism The results of histochemical localization presented here and biochemical studies on glycogen starvation (Siddiqui and Nizami, 1981) suggest that glycogen is the mam energy source in this metacercaria

Acid mucopolysaccharides show a fairly uniform layer around the metacercaria, just below the outer tegumental layer, around the suckers and gonads (Plate I) Previous studies show that acid mucopolysaccharides were localized mainly in the tegument of adult trematodes (Monne , 1959; Nacheva, 1975b), in cercanae (Cheng and Burton, 1966, B e i t o n and Harris, 1967, Rees , 1967) and in the cyst wall of the metacercaria of FascioVa hepafica (D1X o n and M e r c e r, 1964, D1X o n, 1965) and Nanophyetus salmimcola (Porter, 1975) It has been suggested by Harris and Cheng (1973) that the metacercariae which do not form cysts, may possess a thick coating of mucopolysaccharides which might serve some of the functions of the cyst wall formed by other trematode metacercariae Its functional role seems to be concerned with the protection of the worm against the harmful effects of the host's secretions

No information is available on the existence of amyloids in helminth parasites, and their

23

I "S 9S

s

s I •g a >

I s

I

H a s

+ I I + I I

I + + \ +

+ + + + + +

+ + + + +

+ I + + I +

+ + + + +

+ I + + I +

+ + + + +

+ + + + +

I I c

I 3 B

O <

+ + I I + I I

+ + +

+ + + + + + + + + + + +

+ + + + + + + + + + +

+ + + + + + + + + +

+ + + + + + + + + + +

+ 1 I I I + 1 I I I

+ +

+ + + + + + + + +

+ + + + I + I

+ + + + I + I

•SI Iff

I I I + 1

+ +

+ I

I +

+ I + +

+ + + + + + + + + + + + + + +

+ 1 + + + + I + + + + + + +

+ 1 + + + +1 + + + + +

+ + + + + + + + + + + + +

I I + + I I + + + + + + + + +

+ + + + + + + + +

+ + + + + + + + +

+ + I + + I +

K O ^ -S "O o o s ii; a

24

Plate I Fig. 1. Longitudinal section (L. S ) showing glycogen deposition (middle region)

Fig. 2. Diastase treated glycogen — control (middle region) Fig. 3. L. S. showing protein deposition (middle region)

Fig. 4. L. S. showing structural proteins (collagen, myelin and fibrin) by Gomori's trichrome technique (anterior region)

Fig. 5. L. S. showing acid mucopolysaccharides in the tegument (middle region) Fig. 6. L. S. showing lipids deposition (middle region)

presence in moderate amounts have been observed throughout the body except in the tegument where very weak staining for amyloids is obtained (Table 2).

It is difficult to assign any functional role for amyloids in these metacercariae until more detailed information is available on this aspect.

Considerable high concentrations of proteins occur in the parenchyma, musculature, suckers and gonads (Plate I) which suggest that the protein metabolism is probably more active in these tissues. The presence of proteins in the ceca shows that they might be derived from the host during active feeding (Cheng, 1963). Whatever may be the case, accumulation of higher amounts of proteins is in anticipation of enormous egg production.

25

Plate II Fig 7—8. L. S. showing localization of acid phosphatase and control, respectively (anterior region)

Fig. 9—10. L. S. showing localization of alkaline phosphatase and control, respectively (middle region) Fig. 11. L S. showing localization of lipase (middle region)

Fig. 12. L. S. showing localization of non-specific esterases (middle region)

AbreviatioDS used OS = Oral sucker VS = Ventral sucker TE= Tegument P = Parenchyma' T = Testes OV = Ovary V =Vitellaria IC = Intestinal ceca UT = Uterus

Collagen is clearly present in the tegument, suckers (Plate I) and around gonads (Table 2), though it is also present in the parenchyma, musculature, vitellaria and uterine wall but in lesser and varying concentrations. The collagen of different helminths varies in biochemical composition (von Brand, 1973). Histochemically collagen is the least investiga-ted structural protein in trematodes and therefore no specific functional role is attributed to it.

26

The only reasonable functional role available is that the proteins of trematode egg may belong to the collagen group of structural proteins (Bell and Smyth, 1958). It is also likely that the collagen fibres may provide strength to the tissues of the parasite, in order to facilitate active movement inside the host.

Further studies on the collagen synthesis and collagenase are required, however, to ascertain its true functional role in the metacercaria.

Myelin and fibrin localized in the metacercaria and their distribution has been found to be uniform except that myelin is absent in the tegument (Table 2 and Plate I), and might be functioning in association with collagen.

The present histochemical study of lipids shows their significant presence in gonads and suckers (Plate 2 and Table 1). Parenchyma, cecal contents and the tegument possess moderate amounts. The ability of helminths to synthetize fatty acids is very limited (von Brand,1973) . Hence varying amounts of lipids were found by several workers in a number of adult trematodes belonging to different species.

Earlier studies on the localization of lipids in trematodes revealed that the primary site of their presence is the excretory system (Fried and M o o r o n e , 1970; von Brand, 1973; Fried and Pucci , 1976). However, studies on adult Leucochloridiomorpha constantiaedid not show lipids in its excretory system and localized mainly in the intestinal ceca, parenchyma, eggs and suckers (Fried and Pucci , 1976). In the present study the developing vitellaria do not contain significant amounts of lipids. R a o (1959) reported lipids in the excretory system of F. hepatica as well as in the Mehlis' gland but the vitellaria did not contain cholesterol esters, whereas in Gorgodera cygnoides, deposition was observed both in the vitellaria and in eggs, (Schmidt , 1930).

It has been postulated that the higher amounts of lipids, present in parasites which inh ,oit anaerobic environment, were the pro'.' .icts of carbohydrate metabolism (von Brand, 1973; Gupta etai.,1974). The above postujation and also the higher amounts of lipids in testes of this metacercaria (which inhabit a semianaerobic environment) indicates that the growing testes were actively metabolising carbohydrates and their by-products are deposited in the form of lipids. The other view about the occurrence of lipids is that the lipids were synthesized in the parasite body and stored at various regions to meet the energy requirement of various organ system (McManus eta/., 1975).

Histoenzymological studies reveal that lipase is distributed in the tegument, parenchyma, vitellaria and musculature (Table 2 and Plate II). Besides this, biochemical results show that the lipase of this metacercaria is quite active and hormone-sensitive (Siddiq ui and Nizami, 1981). It is possible that lipase might be hydrolysing the triglycerides at or near the body surface and in the intestinal ceca, respectively, ultimately liberating glycerol and free fatty acids, which are being absorbed by simple diffusion for resynthesis of triglycerides, probably by means of a-glycerophosphate pathway (Meyer et a/., 1970).

Among the nuclei acids, DNA is intensely present in the testes and moderately in the ovary, suckers and ceca whereas RNA has been localized in the suckers, parenchyma and comparatively in lesser amounts in the gonads and ceca (Table 2). The presence of nucleic acids in the cecal contents indicates that they might be derived from its host in association with other substances. The presence of DNA and RNA in different structures suggest that possibly these nucleic acids are playing important roles in growth, reproduction and in protein synthesis. Further studies are required to finally assess and elaborate biological functions of

27

the nucleic adds in the protein metabolism of the larval stages in which asexual reproduction is a characteristic feature.

Acid phosphatase gave more intense staining than alkaline phosphatase, which confirms that acid phosphatase is more active than alkaline phosphatase in this metacercaria (S i d d i q u i and Nizami, 1982). Acid phosphatase shows intense reaction in the vitellaria, moderately present in the inner layer of the tegument, parenchyma. The distribution of acid phosphatase in this metacercaria is in accordance with those of other reports on adult trematodes (Sharma, 1976; Smyth, 1976; Gupta and Gupta, 1977). However in adult Ganeo tigrinum the alkaline phosphatase is more intensely present than acid phosphatase (Gupta and Gupta, 1977), which is contrary to our results.

Alkaline phosphatase is present almost exactly at the same site where the acid phosphatase has been observed (Table 2 and Plate II). The only difference is in their intensities. The above results are in accordance with Hal ton (1976b), Sharma and Gupta (1970), Prober t andLwin (1974), Sharma (1976) and G u p t a and Gupta (1977).

The functional role of phosphatase in adult trematodes has been described by different workers as being associated with the breakdown of glycogen and in the active transport of metabolites (von Brand ,1973 ;Dennise ta / . , 1974 ;Lumsden ,1975) and the involvement of alkaline phosphatase in excretion is also well established (von Brand, 1973; T a n d o n and Misra, 1978). The presence of phosphatase in the reproductive organs particularly in the developing vitellaria in the metacercaria under investigation is probably associated with the active metabolism and transport of nutrients for the reproductive organs, which require abundant energy for the synthesis of egg precursors. Such a role for phosphatases has also been attributed in adult trematodes (Halton, 1967b; Y o s h i m u r a and Y o k o g a w a , 1958; Sharma, 1976). Little difference was observed in the distribution of phosphatase in this metacercaria and other previously studied adult trematodes and this leads us to conclude that phosphatases are involved in the energy production of the reproductive organs.

Non-specific esterases have been localized mainly in the regions given in the Table 2. The functional role of esterases in the nervous system is well established (von B r a n d, 1973). The presence of esterases in the tegument (Plate II and Table 2) suggests that like adult trematodes (Nizami et a/., 1977), the esterases have also been secreted and help in the transport of nutrients through the tegument in this non-encysted metacercaria as reported in cestodes (Schwabe efa/., 1961).

Acknowledgements

The authors are grateful to Professor A. H. Siddiqi, Department of Zoology, Muslim University, Aligarh and to Professor J. D. Smyth, Department of Zoology, Imperial College of Sciences and Technology, London for reviewing the manuscript and for their helpful criticism.

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DIXON. K E , MERCER, E (1964) The fine structure of the cyst wall of the metacercana of Fasciola hepatica L O J Microsc Sci, 105 385—389

DRURY, R A B, WALINGTON. E A (1967) Carleton's histological technique London. Oxford University Press, pp 194—213

ERASMUS, D A (1972) The biology of trematodes Edward Arnold. London FRIED. B . MORRONE, L J (1970) Histochemical hpid studies on Echinostoma revolutum Proc

Helmmthol Soc Wash,37 122—123 FRIED, B , PUCCI, D L (1976) Histochemical and thin layer chromatographic analyses of neutral

lipids in Leucochloridiomorpha constantiae (Trematoda) adults Int J Parasitol, 6 479—482 GRIMSTONE, A V . SKAER. R .J (1972) A ^uide book to microscopical methods Cambridge

University Press, pp 54 GUPTA. N A GURAYA.S S .SHARMA.P N (1974) Histochemical observations on the excretory

system of digenetic trematodes Acta Morphol Nederl Scanrf,I2 231—242 GUPTA, S P , GUPTA, R C (1977) Phosphatase activity in Ganeo tigrmum from Rana tigrma 2

ParasitenJc, 54 89—94 H ALTON, D W (1967a) Studies on glycogen deposition in trematoda Comp Biochem Physiol,3

113—120 HALTON, D W (1967b) Studies on phosphatase activity in trematoda J Parasitol, 53 46—54 HARRIS, K , CHENG, T (1973) Histochemical observations on the body surface of Leucochloridio-

morpha constantiae (Trematoda Brachylaemidae) J Parasitof, 59 749—751 LUMSDEN, R (1975) Surface ultrastructure and cytochemistry of parasitic helminths Exp Parasitol,

37 267—339 McMANUS, D P . MARSHALL, I , JAMES, B L (1975) Lipids m digestive gland of Littora sexatibs

rudis (Maton) and daughter sporocyst of Microphallus sitnilis (Jag , 1900) Exp Parasitol, 37 157—163

McMANUS,J F A ,MOWRY,R W (1960) Staining methods histologic and histochemical Paul B HoeberInc New York, pp 229—232

MEYER, F, MEYER, H , BUEDING, E (1970) Lipid metabolism m the parasitic and free living flatworms. Schistosoma mansont and Dugesia dorotocephala Biochim Biophys Acta, 210 257—266

MONNE, L (1969) On the external cuticle of various helminths and their roles m host-parasite relationships A histochemical study Arlc Zooi (Sweden), 12 343—358 ,

NACHEVA L V (1975a) Glycogen deposition in certain trematodes (In Russian) Nauchn Tr Omsk Vet Inst, 31 58—60

NACHEVA. L V (1975b) Histological and histochemical studies of the integument of certain trematodes (In Russian) Nauchn Tr Omsk Vet /nst,31 61—65

NIZAMI,W A,SIDDIOI,A H, ISLAM, M W (1977) Quantitative studies on acetylcholinesterases in seven species of digenetic trematodes Z JParasitenk, 52 275—280

PEARSE,A G E (1961) Histochemistry—theoretical and applied J and A Churchill Ltd , London PORTER C A (1970) A histochemical study of the cyst wall of the metacercana of Manophyetus

satam/co/a (Chaplin) Proc Helmmthol Soc Wash, 37 13—17 PROBERT A J,LWIN,T (1974) Kinetic properties and location of non-specific phosphomonoeste-

rase in subcellular fractions of Fasciola hepatica Exp Parasitol, 35 253—262 RAO K H (1959) Observations on the Mehhs'gland complex in the liver fluke FascioJa hepatica L J

Parasitol, 45 347—351

29

READ, C P, SIMMONS, J E (1963) Biochemistry and physiology of tapeworms Physiol Rev, 43 263—305

REES,F G (1967) The histochemistry of the cystogenous gland cells and cyst wall of Parorchis acanthus Nicoll, and some details of the morphology and fine structure of the cercana Parasitology, 57 87—100

SCHMIDT, W J (1930) CrberdoppelbrechenddLipoideindenDotter-zeilenvonDistomumcygnoides 7eder Zoo/ lahrh , Abt Allg Zool , 47 249—258

SCHWABE, C W , KOUSSA M , ACRA, A N (1961) Host-parasite relationships in echmococcosis IV Acetylcholinesterase and permeability regulation m the hydatid cyst wall Comp Biochem Physiol, 2 161—172 ^

SHARMA, P H (1976) Histochemical studies on the distribution of alkaline phosphatase, acid phosphatase, 5'-nucleotidase and ATPase in various reproductive tissues of certain digenetic trematodes Z Parasitenk , 49 223—232

SHARMA, P N , GUPTA, A N (1970) Histochefflical distribution and functional significance of alkaline phosphatase in the epidermis of certain digenetic trematodes Acta Biol Acad Sci Hung, 21 369—374 *

SIDDIQUI, A A , NIZAMI, W A (1981) Biochemical composition and carbohydrate metabolism of the metacercariae of Chnostomum complanatum (Trematoda Digenea) J Helminthol, 55 89—93

SIDDIQUI, J , NIZAMI, W A (1981) Studies on the lipid metabolism of the metacercariae of Chnostomum comp/anatum (Trematoda Digenea) Int J Parasitol, 11 115—119

SIDDIQUI, A A , NIZAMI, W A (1982) Kinetic and electrophoretic studies on aad and alkaline phosphatases of the metacercariae of Chnostomum complanatum (Trematoda Digenea) / Helmmthol, 56 17—22

SMYTH, J D (1976) Introduction to animal parasitology 2nd Ed Hodder and Stoughton, London THOMAS, R E , GALLICCHIO, V (1967) Metabolism of 14C-glucose by metacercariae of

Chnostomum complanatum (Trematoda) J Parasitol, 53 981—984 TONDON, R E,MISRA, K C (1978) Aad and alkaline phosphatase activities m Fascio/opsis buski

(Lankester, 1857) Odhner, 1902 Ind J Parasito/, 2 145—146 Von BRAND, T (1973) Biochemistry of parasites Academic Press, New York YOSHIMURA, H , YOKOGAWA, M (1958) Physiological studies on the lung fluke III Histochemi-

cal distribution of polysaccharides, nucleic acids and phosphatases in Paragonimus westermam Kerbert, 1878 Jap J Parasitol,! 363—369

rncTOXHMHqecKoe h rHCT03B3HMOjiorinecKoe Hsy eane MerauepKapHeB Clittostomnm complanatum (Trematoda: Digenea)

Bwfloflw

AsTopbi coBepmHjiH FHCTOxHMHiecKoe H rHCT03H3HMonorHqecKoe HsyieHHe MeTaiiepKapneB

Chnostomum complanatum, nojiyHeHHbix H3 rejioBOH nojiocTH npecHOBOflHbix pbi6 Trichogaster fasciatus, onpeaejiaa cjienyiomHe BenjeciBa iJiHKoieH (KapMMH Betra h PAS), KHCJiwe MyKonojiHcaxa-

pHflbi (ajibUHaHOBbiH c h h h h ) , aMHjioHflbi (KOHro KpacHbiii), 6ejiKH (MepKypH-6poM(t)eHonoBbiii c h h h h ) , KOJiJiareH (MajiJiopH aHHJiHH c h h h h ) , m h c j i h h h ( j ) H 6 p H H (Tpofinaa OKpacKa no ToMopH h B a H - r H 3 0 H a

nHKpoc|)yKCHH), jiHnHflbi (cyflaH nepHbiii B), flHK (<I)ejibreH h MeTHjioBbiii s e j i e H b i f l - n H p o H H H ) , A H K

H P H K (MeTHJioBbiii sejieHbiH-nHpoHHH) KHCJiaa H mejioHHaH (JjoctJjaTasbi, jiHnasa h necneuHcJjHMecKHe acTcpasbi onpeflejijMHCb B saMopo-

>KeHHbix cpeaax PeHb Hfler T O > K e o pa3H0M pacnpeAejieHHH h B03M0)KH0ft (})yHKUHOHajibHOH s a f l a i H 3 T H X SHoxHMHHecKHx KOMHOHeHTOB B MeTa6o;iH3Me MerauepKapHeB

C M e K S y j i A j i a M EemA HmaMU

30

Das histochemische und histoenzymologische Sludinm der Metazerkarien Cliaostomum complanalum (Tremafoda: Digenea)

Zusammenfassung

Die Verfdsser ubten em histochemisches und histoenzymologisches Studium der Metazerkarien Chnostomum complanatum aus, erhalten aus der Leibeshohle der Susswassertische Tnchogaster fasaatus, soweit es sich um folgende Stoffe handelt Glykogen (Best Karmin und PAS), sauere Mukopolysacchande (Alzianblau), Amyloide (Kongorot), Eiweisstoffe (Merkur-Btomphenolblau), Kollagen (Mallory-Anilinblau), Myelin und Fibrin (Gomori-Tnchromfarbung und Van Gieson Pikro-fuchsin), Lipoide (Sudan-Schwarz B), DNS (Feulgen und Methylgrun-Pyronin), DNS und RNS (Methyl-grun-Pyronin)

Die sauere Phosphatase, alkahsche Phosphatase, Lipase und unspezifische Esterase wurden in gefrorenen Schnitten festgestelh Man diskutiert auch uber die verschiedentliche Distribution und die moghche funktionelle Aufgabe dieser biologischen Komponenten im Metabolismus der Metazerkarien

S Maqbool Alam WaphA Nizami

Histochemicke a histoenzymologicke studium metacerkirii Cliaostomum complanalum (Trematoda: Digenea)

Suhrn

Autori vykonali histochemicke a histoenzymologicke Studium metacerkarii Chnostomum compla-natum, ziskanych z telovej dutmy sladkovodnych ryb Tnchogaster fasaatus, pokiar ide o nasledujuce latky Glykogen (Bestov karmin a PAS), kysle mukopolysacharidy (alcianovd modrS), amyloidy (konzska cerven), bielkoviny (merkiiro-bromfenolova modra), kolagen (Malloryho anilinova modra), myelin a fibrin (Gomoriho trichromove farbenie a Van Giesonov pikrofuchsm), lipidy (Sudanska £ern B), DNK (Feulgen a metylova zelen-pyronm), DNK a RNK (metylova zelen-pyronin)

Kysla fosfataza, alkahcka fosfataza, lipaza a nespecificke esterazy sa stanovili v zmrazenych rezoch Diskutuje sa tiez o rozdielnej distribucii a moznej funkcnej lilohe tychto biochemickych zloziek V metabolizme metacerkarii

S Maqbool Alam Wajih A Nizami

31

New books

R N Robertson The Lively Membranes Cambndge University Press, 1983 Pp 1—206

In last two decades in helminthology (and in general in parasitology) it is always more of use the new conception of host-parasite interplace which by C P Read et al (1963) is defined as the region of chemical juxtaposition of regulatory mechanisms of both host and parasite involved in determining the nature and thus the outcome of relationship Rapid development of molecular biology and electron microscopy contributes to deepen the knowledge m this sphere The book ofR N Robertsonwill serve for better orientation in new data which through these science branches are introduced also into helminthology although the author did not gather information from works directed to the parasitic way of life or Its manifestations m the host's organism

As the title and content of the book indicates the author deals with behaviour of membrane m hving organisms which have numerous important functions contrary to original conception of their exclusively barrier function (to present from leakage of substances into solution) With such vanety of functions also cohere the differences in the membrane composition although essentially in question is the thickness of two molecules of liquid lipids (Singer and Nicolson, 1972) only Data on properties of biological membranes are described in attractive and instructive way, entirely m i l chapters (with great number of schematic pictures and electrone micrographs) Their more detailed direction is given in the headlines

1 Lively properties — themes and vanations 2 Composition — speaal molecules 3 Structure — cooperative molecules 4 Dynamics — moving molecules 5 Energy transduction — light-energy trapping 6 Energy transduction — oxidation-reduction energy 1 Trans-membrane diffusion — ion earners 8 Transport, absorption and secretion 9 Excited membranes and signal transmission

10 Membrane-bound reactions — hormones, antibodies and synthesis 11 Membranes and evolution For better orientation the subject index is at reader's disposal Besides References at the end of the

book every chapter contains recommended literature to extend reader's scope As seen from aforementioned, the book ofR N Robertson has all attributes of modern handbook

Because of this fact it can serve to workers in various biological branches (physiology, morphology, biochemistry, biophysics, microbiology and plant physiology), including parasitologists It enlarges knowledge from molecular and cellular biology in a way, open to broad arcle of readers

M Breza (Helminthologia, 21, 1984, 1 32)

32

RIVISTA DI PARASSITOLOGIA (IN PRESS)

S T U D I E S ON TlIE I N C I D E I i C E O F I N F E C T I O N , U F £ C Y C L E

A N D HOST S P E C I F I C I T Y O F 0 X Y 3 P I R U R A S T l M d A (NEMATODAI T H E L A Z I I D A B )

BY

$ * MAQBOOL ALAM AND ATHER H . SIDULGlL

S E C T I O N O F P A R A S I T O L O G Y , DEPARTMENT O F ZOOLOCfYj'

A U G A R H MUSLIM U N I V E R S I T y ,

A U G A R H

SUMMARY

T h e i n c i d e n c e o f i n f o c t i o n o f Q . a t u ^ a v a r i e d from ZOfj^-^GlSfjl^ a t d i f f e r e n t p l a c e s o f t h e t y p e l o c a l i t y d u r i n g d i f f e r e n t s e a s o n s * L i f e c y c l e s t u d i e s r e v e a l e d t h a t g r a i n b e e t l e i T r i b o l i u a ^ o p * ) » c o c k r o a c h e s , d u n g b e e t l e s a n d g r a s s h o p p e r s d i d n o t a o t a s i n t e r s m e d i a t e h o s t s * T r i ^ f e r e x p e r i m e n t s c a r r i e d o u t o n c h i c k s s h o w e d t h a t t h e p a r a s i t e f a i l e d t o e s t a b l i s h i n t h e e x p e r i m e n t a l h o s t * i n d i c a t i n g t h a t i t i s q u i t e h o s t s p e c i f i c .

I N T R O D U C T I O N

T h e g e n u s O x v s p i r u r a D r a s c h e i n S t o s s i c h , 1 8 9 7 ( N e m a t o d a t

T h e l a z i i d a e ) p a r a s i t i z e s a l a r ^ ^ g e n u m b e r o f s p e c i e s o f b i r d s *

I n t h e p r e s e n t s t u d y t h e s p e c i e s 0 . s t u m i a D . S * J a i r a J p u r i

a n d S i d d i q i , 1 9 6 7 w a s s e l e c t e d * T h i s p a r a s i t e l i v e s i n t h e

o r b i t a l o a v i t y o f S ^ m , g , m t e X ^ s e o n ,

A c r e d o t h e r e s g i n g i n i a n u s ( L a t h a m ) , a n d F r a n c o l i n u s p o n d i ^ c e r e a n u a *

A l i ( i 9 6 0 ) o b s e r v e d t h a t t h e g e n u s O x v s p i y u r a w a s v w r y h o s t -

s p e c i f i c a n d t h a t u s u a l l y e a c h h o s t h a d i t s o w n n e m a t o d e s p e c i e s *

H o w e v e r n o e x p e r i n « s n t a l o b s o r v a t i o n v / a s m a d e o n t h e h o s t s p e c i f i c i t y

o f £ • s t u m i a a l t h o u g h i t h a s b e e n r e p o r t e d f r o m f o u r d i f f e r e n t

h o s t s * T h e r e f o r e , i t w a s f e l t n e c e s s a r y t o c a r r y o u t s o m e e x p e r i -

m e n t s o n t h e h o s t s p e c i f i c i t y o f t h e p a r a s i t e \ i f t i i ch a p p a r e n t l y

p o s s e s s a l o o s e h o s t s p e c i f i c i t y . A t t e m p t s w e r e a l s o m a d e t o t r a c e

o u t t h e l i f e c y c l e p a t t e r n a n d t h e p o s s i b l e i n t e n a e d i a t e h o s t ( s ) *

T h e f r e q u e n c y a n d t o t a l i n c i d e n c e o f I n f e c t i o n w a s a l s o d e t e r m i n e d *

MATiiRlALS AND Mh;THODS

A number o f b i r d s belongliig to the genus and specioa gtumna

contra w^re c o l l e c t e d Xrom vârlous places of the type l o c a l i t y

during d i f f e r e n t seasons and biôu^t to the laboratory* 'ho P a r a -

s i t o a wero obtained froia behind the n i c t i t a t i n g niombran<a a f t e r

c a r e f u l l y d i s s e c t i n g the eye of the host . These p a r a s i t e s ware

then put i n a P e t r i d i s h containing p h y s i o l o g i c a l sa3.inQ» counted

and prepared for further studies»

Ir^cidencQ of i n f e c t i o n ?

The nurabor of worms per b i r d and per eyewere counted and

recorded to a s c e r t a i n the incidence of i n f e c t i o n .

L i f e cyc le ati idiest

E g g s vrare obtai-ned by d i s s e c t i n g females i n t h e n o r m a l s a l i n e *

developrnent \\fas studied under the microscope o n t e m p o r a r y m o u n t s ,

E g g s were incubated i n thi'ee d i f f e r e n t solut ions* S i © f i r s t w a s

normal sal inep the second normal s a l i n e ©•l?^ formalin (75J25)

a n d the t h i r d normal s a l i n e «001Ja sodium a z i d e s o l u t i o n *

The d i f f e r e n t i n c o c t s experimented upon as IrrtermediatG h o s t s

v;are grain beetle (Tr.itoXiuia sp^, cockroach ( P e r i p l a n a t a s p . ) g r a s s -

hoppers and dung b e e t l e s . These vraro starved f o r t\ô days a a i t h e n

f e d with erabryonated eggs mixed v/ith a little a m o u n t of m o i s t

x ieat f l o u r . After feeding v;ith eggs they \-fQre provided with t h e i r

n o r m a l f o o d . Autopsies o f i n f e c t e d i n s e c t s vrere c a r r i e d o u t a f t e r

5 d a y i n t e r v a l s for 50 days and the contents examined u n d e r t h e

m i c r o s c o p e .

T r a n s f e r e x p e r i m e n t a l

T r a n s f e r Q x p o r i m e n t s \mre c a r r i e d o u t o n 6 d a y o'jld m a l e

c h i c k s ( v i l i t t o l e g l i o r n ) . T h e p a r a s i t e s v / e r e d i r e c t l y t r a n s f e r r e d

f r o m t h e e y e o f S , c o n t r a i n t o t h e e y e o f t h e e x p e r i m e n t a l h o s t

v f i t h t h e h e l p o f a h a i r b r i s t l e . T h e c h i c k s w o r e a u t o p s i e d , t h r o e

a t a t i m e a t 6 h i n t e r v a l u p t o 30 h .

RiSyLTS

I n c i d e n c e o f i n f e c t i o n ?

T h e t o t a l incidence o f in fec t i on in t h e t y p e l o c a l i t y w a s

3 6 # 2 % b u t v a r i e d f r o m i n t h e d i f f e r e n t p a r t s . I n f e c t i o n

d e c r e a s e d d u r i n g Gxtreme h o t weather ( m i d M a y - J u n e ) a n d a g a i n

i n c r e a s e d t h e r e a f t e r w i t l i t h e o n s e t o f r a i n y s e a s o n ( T a b l e I ) «

T h e m e a n n i x m b e r o f p a r a s i t e s o b t a i n e d f r o m e a c h b i r d v a r i e d

b u t n e v e r e x c e e d e d 5 p e r b i r d a n d t h r e e p e r e y e .

L i f e c y c l e s t u d i e s t

T h e e g g s o b t a i n e d f r o m t h e d i s t a l p a r t o f u t e r i w e r e f o i u i d

t o c o n t a i n l U l l y f o r m e d l a r v a e , g e n e r a l l y i n t h e f o r m o f t h e

f i g u r e o f * 8 » ( F i g , I B ) » E g g h a t c h i n g a n d e m e r g e n c e o f l a r v a e t o o k

p l a c e ^ v i t r o a f t e r 6 h i n t h c ' n o r m a l s a l i n e o n l y . T h e l a r v a e s o

h a t c h e d d i d n o t s h o w d i i t i n ^ s h a b l e c h a r a c t e r s t i c f e a t u r e s ( F i g , 1 A ) ,

T h e e g g s i n c u b a t e d i n t v / o o t h e r s o l u t i o n s d i d n o t s h o w a r ^ c h a n g e .

T h e a u t o p s y o f e x p e r i m e n t a l i n t e r m e d i a t e h o s t s r e v e a l e d t h a t

t h e y w e r e h a r b o u r i n g n e i t h e r t h e e g g s n o r a n y o f t h e d e v e l o p m e n t a l

s t a g e s .

Transfer experiments»

T h e p a r a s i t e s c o u l d n o t b e t r a c e d t h r o u g h o u t t h e a l l r d e n t a r y

o a n a i o f fchc oxporltnGntal h o s t a f t e r 6 / h b u t t h e e g g s w e r e p r e s e n t

i n v a r i o u s r e g i o n s o f t h e d i g e s t i v e t r a c t ( T a b l e I I ) ,

DISCUSSION

V a r i a t i o n i n t h a i n c i d e n c e o f i n f e c t i o n a t d i f f e r e n t

p l a c e s o f t i i e t y p e l o c a l i t y d u r i n g d i f f e r e n t s e a s o n s m a y p r o b a b l y

b e d u e t o n o n - a v a i l a b i l i t y o f s u i t a b l e i n t e r m e d i a t e h o s t ( s ) a n d

a l s o d u e t o t h e d e s s i c a t i o n o f t h e t h i n w a l l e ( 3 e g g s ,

^Die f a c t t h a t t h e i n f e c t a d b i r d s w e r e n e v e r f o u n d t o h a r b o u r

m o r e t h a n 5 p a r a s i t e s a t a t i m e and maxiiaum 3 i n o n e e y e l a a y b e

s u ^ ^ e s t l v e o f t h e l e s s n u t r i t i v e v a l u e o f t i i e e y e or l a c k o f

a v a i l a b i l i t y o f s p a c e t o l i v e i n , M o r o o V e r , i f t h e n u m e r i c a l

s t r e n g t h o f t h e p a r a s i t e i n c r e a s e d i t v j o u l d i n t r e f e r e i n t h e n o r m a l ^ \

f u n c t i o n i n g o f t h e e y e . T h i s m i g h t r e s u l t i n t h e d e a t h o f t h e h o s t .

I t i s o b v i o u s l y t o t h e d i s a d v a n t a g e o f t h e p a r a s i t e t o c a u s e d e a t h

o f i t s hozt, f o r i n GO d o i n g i t d e s t r o y i t s e l f , .

T h e d e v e l o p m e n t a n d h a t c h i n g o f l a r v a e i n n o r m a l s a l i m

i n d i c a t e s t h a t t h e p a r a s i t e r e l e a s e s e g g s , v d t h f u l l y d e v e l o p e d

l a r v a e i n t h e h o s t b o d y » v A i l c h t h e n u l t i m a t e l y p a s s e s o u t v f l t h t h e

f a e c e s o f t h e h o s t .

T h e g e n u s O x v s p i r u r a h a s b e e n d e ' ^ b e d t o b e l ^ g h l y h o s t

s p e c i f i c ( A l l , I 9 6 0 ) , T r a n s f e r e x p e r i m e n t s c a r r i e d o u t o n 6 d a y o i d

c h i c k s a n d t h e f a i l u r e o f t h e p a r a s i t e t o e s t a b l i s h i n i t m a y

s u g g e s t t h a t s t u r n i a i s s e l e c t i v e l y h o s t s p e c i f i c * T h i s a s s u m p *

t i o n i s , h o w e v e r p b a s e d o n t h e f a c t t h a t t h i s p a r a s i t e h a s b e e n

r e a r d Q d f r o m f o u r d i f f e r e n t s p e c i e s o f b i r d s i n c l u d i n g a g a l l i f o r i Q

( F r a n c o l i n u s p o n d i c e r e o n u s ) t o ^ A i l c h g r o u p t h e c h i c k s a l s o b e l o n g .

T h e p r e s e n c e o f t h e e g g s i n t h e a l i m e n t a r y c a n a l i n d i c a t e d

t h a t t h e p a r a s i t e m i g r a t e a i n t o t h e d i g e s t i v e t r a o t « m o s t p r o b a b l y

t h r o u ^ t h e n a s o p h a r y n g e a l p a s s a g e , m s d i g e s t e d t h e r e a M e l i »

n i n a t e d w i t h t h e f a e c e s .

A n o t h e r p o i n t p r e s u m e d d u r i n g t h e s t u d y o f h o s t s p e c i f i c i t y

w a s t h a t t h e p h y s i o l o g y o f t h e e y e s h o u l d b e s i m i l a r i n m o s t c a s e s ,

H o v j o v e r f f a i l u r e o f t h e p a r a s i t e t o e s t a b l i s h i n t h e © y e o f t h e

0 3 i 5 ) e r i m e n t a l h o s t m a y p r o b a b l y i n d i c a t e t h a t s o m e l A i y s i o l o g i c a l

d i f f e r e n c e s d o e x i s t i n t h e e y e s o f d i f f e r e n t b i r d © w h i c h h i n d e r s

t h e e . ' j t a b l i s h m e n t o f t h e p a r a s i t e . T h i s i s q u i t e c o n t r a r y t o t h e

c a s e o f t h e t r e m a t o d e P h i l o p h t h a l a m u a s p , % A i i c h i ^ e n t r a n s f e r r e d

f r o m i t s d e f i n i t i v e h o s t t o a n y e x p e r i m e n t a l a n i m a l , e a s i l y e s t a b * -

l l s h e d i n t h e e y e o f t h e n e w h o s t ( M c M i l l a n & M a c y » 1 9 7 2 ) « T h e s e

t w c o n t r a d i c t o r y r e s u l t s m a y b e d u e t o t h e d e g r e e o f h o s t

s p e c i f i c i t y o f t h e c o n c e r e n o d p a r a s i t e s , 0 , s t u m i a i s m o r e h o s t

s p e c i f i c a n d t h e r e f o r e f a i l s t o d e v e l o p i n d t h e r e x p e r i m e n t a l

h o s t s TfrtiilG H i i l o i p h t h a l a m u s s p , i s c a p a b l e o f a d a p t i n g i t s e l f i n

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A f e w e g g s I n t h e g i z z a r d c o n t e n t s

£ g g s p r e s e n t i n t h e i n t e s t i n a l c o n t e n t s

A f e w d e f o r m e d e ( p e r s e n t I n t h e r e c t a l c o n t e n t s

A l l , S . M , ( 1 9 6 0 ) , O n s o m e n e w s p e c i e a o f t h e g e n u s O x v s p l r u r a

. f r o m b i r d s I n H y d e r a b a d , A n d h r a P r a d e s h , I n d i a . J .

l l e l m i n t h o l . , 3 4 s 2 2 4 - 2 4 2 ,

J d i i i A ^ p u r i , D » S , a n d S i d d i q i , A J U . ( 1 9 6 7 ) . A r a v i e w o f t h e g e n u s

Q ^ y s p l r u r . a D r a a c n e i n S t o a s i c h , 1 8 9 7 ( W e s n a t o d a i

T h e l a s i i d a e ) w i t h d e s c r i p t i o n o f f o u r t e e n n e w s p e c i e s ,

J . H G l m i n t h o l . 4 l : 3 3 7 - 3 6 3 .

M c l i l l a n , T . A . a n d M a c y , i l . . v . ( 1 9 7 2 ) . T h e l i f e c y c l e o f

P h i l o p h t h a l a m u a m e f i : a l u r u s ( C o r t , 1 9 1 4 ) ( T r e m a t o d a s

P h i l o p h t h a l a i n i d a e ) Jn W e s t e r n Oregon. J . P a r a a i t o l *

5 6 » 2 2 .

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