CHEMICAL COMPOSITION OF EGG SHELL IN MONIEZIA EXPANSA (RUDOLPHI, 1805) A. Gupta, S. K. Srivastav* and V. Gupta *P.G. Department of Zoology, Shia P.G. College Lucknow. Department of Zoology, University of Lucknow, Lucknow

ABSTRACT: The cestode Moniezia expansa possesses very small vitelline gland and therefore has very thin egg-shell. The chemical composition of the egg-shell of M. expansa has been investigated using histochemical techniques. There are three precursors of egg-shell viz. phenolase, phenols and Proteins. Of these precursors, only protein precursors is found to be present in M. expansa and phenols and phenolase are found to be absent The presence of basic protein ,tyrosine ,keratin, protein containing disulphide (-SS-) and (-SH-) linkages indicate the egg-shell to be of keratin-type. Arginine, elastin, and lipid were absent. Besides protein, glycogen and lipids were also present.

Key words: EGG SHELL, MONIEZIA EXPANSA, HISTOCHEMISTRY, KERATIN. INTRODUCTION: Eggs of helminths can be divided into two groups: group one, those from helminths whose hosts have aquatic association and the larvae hatch in the external environment example trematodes and pseudophyllidean cestodes and group two, those from helminths whose hosts have terrestrial associations and the larvae hatch with in the intermediate or definitive host example cyclophyllidean cestodes. Therefore, the egg shells in these two groups vary in

their degree of resistance to external conditions and chemical nature of the egg-shells. The present study is undertaken to investigate the chemical composition of egg shell of Moniezia expansa, a cyclophyllidean cestode belonging to group two by various histochemical tests and its importance in further development of egg.

MATERIALS AND METHODS Moniezia expansa were collected from the sheep intestine brought from the local abattoir. The worms were washed thoroughly in normal saline. The gravid proglottids were fixed in bouins fluid, carnoy’s fixative10% formalin and 70% alcohol, dehydrated, cleared and embedded in paraffin wax. Sections of 6-8 µm thickness were cut on rotatory microtome and various histochemical tests were performed. The histochemical tests were also directly applied on eggs which were taken out by teasing the gravid proglottids. Various histochemical tests were adopted. Tests for proteins was done by Xanthoproteic test (Pearse, 1953), Biuret Test (Srivastava, 1978), tests for basic protein by Aqueous bromophenol blue method (Mazia, Brewer And Alfert, 1953), Ninhydrin Schiff's test (Pearse, 1968) test for Arginine containing proteins by

Sakaguchi's reaction (Baker’s1947 modification in

Pearse,1968),test for Tyrosine containing proteins by Millon's Test (Bensley and Gersh modification as in Pearse, 1968), test for Elastin by Verhoeff's Elastin Stain (Pearse, 1968), test for Collagen by Mallory's Triple stain (McManus And Mowry, 1963),test for proteins containing Sulphydrl group (-

SH-) by ferric ferricyanide method (Chevremont and Frederic, 1943 as in Pearse, 1968), test for proteins containing Disulphide group (–SS–) by Thioglycollate-Ferric Ferricyanide method (Adams, 1956 as in Pearse,1968). Test for Keratin by Heidenhain's iron haematoxylin test (Carleton And Leach, 1938). Test for Phenolase by Catechol test (Johri, L.N. and Smyth, J.D., 1956). Test for Phenols by Argentaffin reaction (Bell and Smyth, 1958), Chromaffin Reaction (Lewis, 1962), Ferric Chloride Test (Srivastava, 1978), Sodium Iodate Test (Srivastava, 1978) , Tests For Glycogen Best's Carmine Method (Best, 1906 as in Pearse, 1968) ,Periodic Acid Schiff's Reaction (After McManus as in Pearse, 1968), Test For Lipid Sudan Black B Test (After McManus, 1946, as in Pearse, 1968). RESULTS The results of various histochemical tests performed on the egg-shell of Moniezia expansa have been summarized in Table 1. The egg-shell showed the absence of phenolase and phenols. It was strongly positive for tests for proteins and basic proteins, moderately positive for tyrosine Arginine was found to be absent. To find out the actual chemical nature of the egg-shell, tests for other structural proteins were also employed. Elastin and collagen have not been observed. The presence of keratin, proteins containing sulphydryl (-SH-) group and disulphide (-SS-) group has been noticed in the egg-shell of Moniezia

expansa.

Test for glycogen and lipid were also positive in the egg-shell. The absence of phenolase and phenols and the presence of protein, basic protein, tyrosine, keratin, protein containing –SH- and –SS- groups shows the egg-shell of Moniezia expansa to be of keratin type. Table:1 Results of various histochemical tests performed on the egg-shell expansa

of Moniezia

Staining reaction for

Test adopted

Egg shell

Phenolase

Catechol reaction

–

Phenols

Chromaffin reaction

–

Argentaffin reaction

–

Sodium iodate test

–

Ferric chloride test

–

Xanthoproteic test

+++

Biuret test

+++

Aqueous bromophenol blue technique

++

Ninhydrin Schiff's reaction

++

Tyrosine

Millon's reaction

++

Arginine

Sakaguchi reaction

–

Elastin

Verhoeff's stain

–

Collagen

Mallory's triple stain

–

Keratin

Heidenhain's iron-haematoxylin test

+++

Proteins having –SH– linkages

Ferric ferricyanide test

+++

Proteins having –SS– linkages

Thioglycollate-ferric ferricyanide test

+++

Glycogen

Best's carmine test

+

Periodic acid Schiff's reaction

+

Sudan Black B test

+

Protein

Basic protein

Lipid

(+++) Intense reaction; (++) Moderate reaction; (+) Present; (–) Absent

DISCUSSION Various histochemical tests were employed to find out the chemical composition of egg-shell of Moniezia expansa. The results from the present study show that out of the three precursors of egg shell, phenols and phenolase were absent and only protein precursor was present. The egg-shell was found to contain basic proteins, tyrosine, keratin, proteins having –SS– and –SH– linkages. Thus, the egg-shell was of keratin type and not of quinone-tanned (sclerotin) type. Arginine, elastin and collagen were absent. These results are in accordance with those reported previously for the same parasite (Hoy and Clegg, 1967 as referred, to by Clegg and Smyth, 1968; Arfin and Nizami, 1986). They observed a positive reaction for disulphide linkage showing the capsule or shell to be of keratin type of protein. Moreover, the available literature on the nature of the embryophore in cyclophyllidean cestodes reveals that it is keratinized e.g. Dipylidium caninum (Pence,1967), Hymenolepis diminuta (Pence, 1970; Lethbridge, 1971), Multiceps smythi (Johri, 1957); Taenia hydatigena, T. ovis and T. pisiformis (Morseth, 1966). Besides protein, glycogen and lipid were also found in the egg-shell of Moniezia expansa. Johri (1957) found the absence of polysaccharides and lipids in the embryophore of Multiceps smythi. The glycogen and lipids were reported for the first time in the egg-shell of M. expansa. Most of the trematodes and pseudophyllidean cestodes have all the three egg-shell precursors i.e. phenolase, phenols and proteins. Thus, they

have the sclerotin or quinone-tanned type of egg-shells (Smyth and Clegg, 1959). Most trematodes and pseudophyllidean cestodes have well developed vitellaria and lay their eggs into water and the larvae hatch in external environment. But the case differs in many cyclophyllidean cestodes including Moniezia expansa in possessing small, compact vitelline gland and the gravid proglottids are detached from the parasites (apolysis) and excreted in the host faeces and later on eggs are released from the proglottids on land from where they are taken by intermediate host for the further development. Thus, there must be variation in egg-shell of Moniezia expansa from that of trematode and pseudophyllidean cestodes in their degree of resistance to external environment as well as in chemical nature. It is found in present study that in Moniezia expansa, the egg-shell is keratin type unlike most of trematodes where it is sclerotin or quinone tanning type. Smyth And Clegg (1959) suggested that probably the sclerotin capsule of trematodes and pseudophyllidean cestodes protect the embryo in egg during lengthy embryonation period in water. In many cyclophyllidean cestodes, the eggs are often released from proglottids on land where they may withstand desiccation and mechanical damage. Thus, it may be possible that the keratin type of protein retards desiccation of the onchosphere on land more successfully than sclerotin. Sclerotin is very resistant to hydrolysis, not easily digested by the intermediate or final hosts on ingestion. The taeniid embryophore which is of keratin type (Johri, 1957) is easily broken down by mammalian digestive secretions (Silverman, 1954a).

It may be possible that keratin type of egg-shell in Moniezia expansa is helpful in retarding desiccation of eggs released from proglottids on land and it may be easily digested by the intermediate hosts, the mites and thus allowing further developmental processes. Trematodes and pseudophyllidean cestodes having well developed vitellaria possess thick sclerotin egg capsule (Smyth and Clegg, 1959) whereas cyclophyllidean cestodes have small vitelline glands, small vitelline cells with small nuclei surrounded by a thin layer of cytoplasm and a large vesicle. Such types of vitelline cells have been observed in a number of cyclophyllidean cestodes (Bischoff, 1913; Venard, 1938; Ogren, 1956; Johri, 1957; Rybicka, 1964). When these vitelline cells enter the uterus, they loose the vesicle and discharge reserve material which accumulates in the thin membranous capsule formed around the embryo. Further, the histochemical studies reveal that these cells contain acid mucopolysaccharide glycogen or acidophilic proteins (Smyth and Clegg, 1959; Loser, 1967; Smyth, 1969, 1976) contribute to the transport of material for capsule formation and accumulate nutritive reserves. This indicates that the vitelline cells play an important role in egg-shell formation. Therefore, M. expansa due to the presence of small vitelline glands have thin shell. It matures during the passage through the uterus where a glandular secretion is deposited on the outer surface (Clegg and Smyth, 1968; Fairweather and Threadgold, 1981). Thus, the present and previous studies lead to conclude that egg-shell in Moniezia expansa is keratinized and different from quinone-tanned egg-shell of trematodes and pseudophyllidean cestodes.

ACKNOWLEDGEMENT Financial assistance (Junior research fellowship) provided by council of scientific and industrial research (C.S.I.R) to authors for this work.

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Photographs of Egg-shell Composition in Moniezia expansa (Rudolphi, 1805).

Eggs of Moniezia expansa showing the presence of basic protein in the egg-shell with aqueous bromophenol blue technique.

Eggs of Moniezia expansa showing the presence of tyrosine in the egg-shell with Millon's reaction.

Eggs of Moniezia expansa showing the presence of SS-group containing protein in egg-shells with Thioglycollate-ferric ferricyanide test.

Eggs of Moniezia expansa showing the presence of keratin in eggshell with Heidenhein's iron-haematoxylin test.

T.S. of gravid proglottid of Moniezia expansa showing the presence of SH-group containing protein in the egg-shell with ferric ferricyanide test.

L.S. of gravid proglottids of Moniezia expansa showing the presence of lipid in egg-shell with Sudan black B test.