A Technique for the Histochemical Demonstration of Polyphenol Oxidase and its application to Egg-shell Formation in Helminths and Byssus Formation in Mytilus

1. The distribution of polyphenol oxidase in quinone-tanning systems may be demonstrated in frozen-dried sections by incubation in 0.2 per cent, aqueous catechol at 400° C. for 15-60 minutes. A red colour develops at the enzyme site. 2. The evidence for the view that the egg-shell in trematodes, in certain cestode groups, and in turbellarians, is a quinone-tanned protein secreted by the so-called ‘vitelline’ glands, is summarized. The ‘vitelline’ cells, in addition to giving positive reactions for proteins and phenols, give a strongly positive reaction with the catechol polyphenol oxidase test. 3. The catechol technique may also be applied to whole helminths fixed in 70 per cent, alcohol, and serves as a useful whole mount stain for the shell-producing regions of the female genitalia. 4. In Mytilus the catechol technique reveals the presence of polyphenol oxidase in an ‘upper’ or enzyme gland in the foot. 5. It is suggested that in Mytilus the byssus is formed from a phenolic protein secreted from the phenol gland, which on contact with polyphenol oxidase can undergo ‘auto-quinone tanning’.

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A histochemical approach to the study of helminth morphology

Parasitology ◽

10.1017/s003118200002638x ◽

1956 ◽

Vol 46 (1-2) ◽

pp. 107-116 ◽

Cited By ~ 97

Author(s):

L. N. Johri ◽

J. D. Smyth

Keyword(s):

Malachite Green ◽

Polyphenol Oxidase ◽

Shell Material ◽

Basic Proteins ◽

Histochemical Methods ◽

Whole Mount ◽

Quinone Tanning ◽

Phenol Blue ◽

Egg Shell ◽

Whole Mounts

1. The egg-shell in many trematodes and pseudophyllidean cestodes is a quinone-tanned protein; and histochemical methods for polyphenol oxidase or for phenols may be used to demonstrate regions producing or containing shell material (i.e. the vitellaria, its ducts and the uterus) in whole mount preparations.2. Specimens so treated may be further stained by routine whole mount methods. Gower's carmine is particularly recommended.3. 70% alcohol was found to be the best general fixative for these methods.4. These histochemical methods—with certain modifications—may also be used for demonstrating the egg-shell producing regions in sections.5. Because of their affinity for basic proteins, bromo-phenol-blue and malachite green are suitable for staining these regions in sections. These stains are unsuitable for whole mounts.6. There is some evidence to suggest that all trematodes and pseudophyllidean cestodes do not form and harden their egg-shell by quinone tanning; consequently, some forms may give negative or unsatisfactory results with these methods.

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Studies on the structure of the female reproductive system and egg-shell formation inAspiculuris tetrapteraSchulz, (Nematoda: Oxyuroidea)

Parasitology ◽

10.1017/s003118200008272x ◽

1964 ◽

Vol 54 (4) ◽

pp. 699-719 ◽

Cited By ~ 19

Author(s):

A. O. Anya

Keyword(s):

Reproductive System ◽

Shell Formation ◽

Female Reproductive System ◽

High Concentration ◽

Classical Study ◽

Quinone Tanning ◽

Phenolic Substances ◽

Egg Shell ◽

Exogenous Origin ◽

Aspiculuris Tetraptera

The histological anatomy of the female reproductive system of an oxyuroid nematode,Aspiculuris tetraptera, Schulz, has been described.The process of egg-shell formation in this animal has been followed in detail while the structural and chemical characteristics of the egg-shell have been studied by histochemical and other methods. It is shown that there are three layers: a lipoprotein layer, a ‘chitinous’ layer and the so-called vitelline (glycosidal) membrane. Evidence is presented for the exogenous origin of the lipoprotein layer: this being formed by the cells of the upper uterus which are shown to be secretory.The question of quinone-tanning in the egg-shell ofA. tetrapteraand in other oxyuroids and ascarids is considered. It is shown that neither a polyphenol oxidase nor a high concentration of phenolic substances (apart from protein tyrosine) exists in this system. The significance of these and other observations is discussed in relation to the mechanism of tanning as elucidated in insects and trematodes.I have to acknowledge with gratitude the support of many during these investigations. To Dr P. Tate for his encouragement and provision of facilities at the Molteno Institute; to Dr D. L. Lee, for much useful discussion and permission to refer to some of his unpublished electron micrographs of nematodes; to Professor J. D. Smyth, who kindly read through the manuscript; to the Cambridge Philosophical Society for a grant that made possible the translation of Fauré-Frémiet's classical study onAscaris;and to the Department of Technical Cooperation for financial assistance.

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Studies on Penetrocephalus ganapatii, a new genus (Cestoda: Pseudophyllidea) from the marine teleost Saurida tumbil (Bloch)

Parasitology ◽

10.1017/s0031182000025257 ◽

1960 ◽

Vol 50 (1-2) ◽

pp. 155-163 ◽

Cited By ~ 7

Author(s):

K. Hanumantha Rao

Keyword(s):

Bay Of Bengal ◽

New Genus ◽

Shell Formation ◽

Marine Teleost ◽

Female Reproductive System ◽

Form Part ◽

Vitelline Cells ◽

Histochemical Tests ◽

Egg Shell ◽

Egg Capsule

1. The character of the scolex, the long filamentous neck and the morphology of the proglottids are features which justify the establishment of a new genus, Penetrocephalus, to accommodate the pseudophyllid species P. ganapatii (= Both-riocephalus ganapatii (Rao, 1954), B. penetratus (Subhapradha, 1955)) from the marine teleost Saurida tumbil Bloch, off Waltair, Bay of Bengal.2. The anatomy of the female reproductive system is described in detail, and a preliminary interpretation of the histochemistry of egg-shell formation is given.3. On the basis of histochemical tests it is suggested that Mehlis's gland, which surrounds the cellular ootype, secretes a phospholipid-like material which may aid in the release of shell precursors from the vitelline cells, but it does not form part of the shell. It appears also to be responsible for the synthesis of PAS-positive yolky matter found in these cells in the egg capsule. The functions of Mehlis's gland in helminths are discussed.4. In a majority of helminths where the egg-shell is a quinone-tanned protein, it is considered that it is solely if not entirely derived from shell globules in the vitelline cells.

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The problem of Mehlis's gland in helminths with special reference to Penetrocephalus ganapatii (Cestoda: Pseudophyllidea)

Parasitology ◽

10.1017/s0031182000025440 ◽

1960 ◽

Vol 50 (3-4) ◽

pp. 349-350 ◽

Cited By ~ 11

Author(s):

K. Hanumantha-Rao

Keyword(s):

Special Reference ◽

Positive Reaction ◽

Periodic Acid ◽

Gland Secretion ◽

Periodic Acid Schiff ◽

Vitelline Cells ◽

Egg Shell

1. Mehlis's gland secretion in trematodes and pseudophyllidean cestodes may be a lecithin-like phospholipid.2. After enzymic degradation of the lecithin into lysolecithin the release of egg-shell precursors from the vitelline cells is effected.3. Soon after the completion of the egg-shell the lysolecithin may be reconverted into lecithin in vitelline cells of Penetrocephalus ganapatii, thus accounting for the periodic acid-Schiff positive reaction.

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Cytological and histochemical criteria for evaluating development of trematodes and pseudophyllidean cestodesin vivoandin vitro

Parasitology ◽

10.1017/s0031182000021132 ◽

1958 ◽

Vol 48 (1-2) ◽

pp. 131-148 ◽

Cited By ~ 54

Author(s):

E. J. Bell ◽

J. D. Smyth

Keyword(s):

Cell Multiplication ◽

Developmental Phase ◽

Shell Formation ◽

Fresh Tissue ◽

Final Maturation ◽

Whole Mount ◽

High Mitotic Activity ◽

Egg Shell

1. Maturation of the plerocercoid of the pseudophyllidean cestodeDiphyllobothriumsp. and the metacercaria of the strigeid trematodeDiplostomum phoxinifollow a similar pattern, which may be divided into the following phases: (1) cell multiplication; (2) segmentation or body shaping; (3) organogeny; (4) early gametogeny; (5) late gametogeny; (6) egg-shell formation and vitellogenesis; (7) oviposition.2. Cytological or histochemical criteria have been developed by means of which each developmental phase may be recognized. Developmentin vitromay be considered comparable with that occurringin vivoif the same phases are completed with the same incubation time and without the appearance of cytological abnormalities.3. The cell multiplication phase is characterized by high mitotic activity. This activityin vitrocan be evaluated by incubating larvae after 24 hr. culture for a standard time (5 hr.Diphyllobothrium; 4 hr.Diplostomum) in 10−4colchicine and comparing the number of mitoses seen in a defined area of an aceto-orcein squash with those found in tissue maturedin vivofor the same period. Use of this criterion enables the growth-producing properties of a medium to be assessed after 24 hr. culture.4. Phases 2–4 are characterized by the appearance of the tubular genitalia, the rudiments of which are readily recognized in aceto-orcein squashes or whole mount preparations.5. The presence of spermatids and mature spermatozoa (phases 4–5) may be detected in aceto-orcein squashes or in fresh tissue teases. The quantity and activity of spermatozoa also serve as important criteria.6. Recognition of phase 6, egg-shell formation, is carried out by means of diazo reagents which give orange or orange-red reactions with the phenolic egg-shell precursors in the vitellaria, as seen in whole mount preparations. Abnormal egg-shell development is reflected in the failure of vitellaria to give a normal diazo reaction.7. The appearance of eggs which are macroscopically normal constitutes the final maturation phase. Early detection of eggs (especially abnormal ones) is facilitated by the use of the diazo reaction or the catechol test for polyphenol oxidase.8. Embryonation and hatching of eggs to produce morphologically normal infective coracidia or miracidia serve as a final criteria.9. Criteria for the maintenance of the adult condition are also considered.

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Shell gland and egg-shell formation in Fasciola hepatica L.

Parasitology ◽

10.1017/s0031182000018345 ◽

1953 ◽

Vol 43 (1-2) ◽

pp. 88-93 ◽

Cited By ~ 8

Author(s):

H. K. Yosufzai

Keyword(s):

Fasciola Hepatica ◽

Shell Gland ◽

Shell Formation ◽

Vitelline Cells ◽

Egg Shell

1. The shell gland of Fasciola hepatica produces an extremely hyaline secretion. The egg-shell is formed from the hyaline secretion.2. As the eggs travel through the uterus, the shell is temporarily reinforced from the inside by vitelline granules. The vitelline granules are released when the vitelline cells enter the vitelline reservoir.3. Views regarding the nomenclature of the shell gland are discussed.4. The presence of an elliptical chamber in the shell gland is described. In this chamber, oocytes, vitelline granules and vitelline cells are mixed together, and here the secretion of the shell gland is released and the egg-shell is formed.5. The arrangement of the cellular constituents within the egg is effected by the elliptical chamber.

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