The Cytoplasmic Inclusions of Nyctotherus macropharyngeus: Histochemical Studies

Richardson and Horning (1931) described polymorphic ‘Golgi bodies’ in N. cordiformis, varying in shape from short rods to twisted filaments. In the same species Patten (1932) described certain twisted, snake-like objects as Golgi bodies. Khajuria (1950) gave the same name to granules in the cytoplasm of N. macropharyngeus.

Horning (1927) and Richardson and Horning (1931) described the mitochondria of N. cordiformis as rod-shaped; Patten (1932) described them as spherical granules. Khajuria (1950) described those of N. macropharyngeus as globular and rod-shaped.

‘Vegetative granules’ have been described in Nyctotherus by the authors named above. They appear as transparent spheres in the living organism, and are unstained by iron haematoxylin in fixed preparations. In the present paper they will be called carbohydrate bodies.

As far as the author is aware, no paper dealing with the histochemistry of the cytoplasmic inclusions of Nyctotherus is available. The purpose of the present work was to investigate, both morphologically and histochemically, the cytoplasmic inclusions of N. macropharyngeus.

Living specimens were examined in 0·85% sodium chloride solution by phase-contrast microscopy. Janus green B, neutral red, and other vital dyes were also used.

Specimens were placed in 2% osmium tetroxide solution and examined without further treatment.

The following fixatives were used for specimens that were to be sectioned subsequently: Lewitsky (strong Flemming without acetic), Regaud, Helly, Bouin, Carnoy. Paraffin sections were cut at 5μ. They were stained either with iron haematoxylin (0·5% haematoxylin) or with acid fuchsine (Cain, 1948b).

Details of the histochemical tests used are given in the appendix (p. 521).

In addition to the contractile vacuoles and food vacuoles, there are three distinct types of cytoplasmic inclusions: lipid bodies, mitochondria, and carbohydrate bodies.

These are distributed at random in the medulla. Each appears to be duplex, consisting of externum and internum. The duplex structure is seen in the living organism and also after treatment with osmium tetroxide solution (fig. 1, A) and with Sudan black (fig. 1, B). The externum is darkened by the two latter techniques, while the internum remains clear. The externum appears crescentic in Sudan black preparations, and also in preparations coloured by mercuric bromophenol blue (fig. 1, c). The latter result suggests the presence of protein in the externum. Ciaccio’s ‘unmasking’ technique (see Gupta, 1958), followed by colouring with Sudan black, gives the same crescentic appearance as is seen in ordinary Sudan black preparations. The internum evidently contains no lipid; indeed, it did not give a positive response to any of the histochemical tests that were tried.

These are rod-shaped. They are arranged in a peripheral layer just below the pellicle, and some are scattered also in the medulla. They are stained by mercuric bromophenol blue (fig. 1, c) and by acid fuchsine (fig. 1, D); they are also darkly stained by iron haematoxylin in material fixed in Lewitsky, Regaud, or Helly. They stain supervitally with Janus green.

Each of these is composed of numerous small spheres (fig. 2). The superficial part of each sphere is PAS-positive. The carbohydrate bodies give no positive response to tests for proteins or lipids, and are not stainable by neutral red during life.

The small spheres develop in close association with mitochondria (fig. 1, D). As each sphere grows, the associated mitochondrion begins to lose its affinity for acid fuchsine. Ultimately, a number of spheres coalesce to form larger bodies.

The evidence suggests that the externum consists of neutral lipid, probably triglyceride, and protein. The composition of the internum is unknown.

In Opalina ranarum the lipid bodies consist of triglyceride and lipoprotein; in O. scalpriformis, of lipoprotein only (Dutta, 1958). These two species and N. macropharyngeus live in the same environment, as gut parasites of frogs and toads, but their lipid bodies differ considerably in chemical composition.

The lipid bodies correspond to the so-called ‘Golgi bodies’ of earlier workers (Richardson and Horning, 1931; Patten, 1932; Khajuria, 1950).

The carbohydrate bodies or ‘vegetative granules’ were supposed by Horning (1927), Richardson and Horning (1931), and Khajuria (1950) to function as nutritional stores. It is possible that they develop under the influence of enzymes produced by mitochondria, as suggested by these authors.

This work was carried out in the Department of Zoology, Panjab University, Hoshiarpur, under the supervision of Prof. Vishwa Nath, Head of the Department, to whom I am greatly indebted for allowing me the necessary laboratory facilities. I am thankful to Mr. Brij L. Gupta, technician to the Department, for some useful help and suggestions.