The Distribution of Vitamin C in Nyctotherus Cordiformis Ehrenberg, Opisthioglyphe Ranae Frölich, and Toxocara Canis Werner

The possibility that internal parasites may play a part in the vitamin metabolism of the host was first suggested by Roskin & Nastiukova (1941). Working on Trypanosoma brucei in guinea-pigs, these workers demonstrated the presence of vitamin C granules in the cytoplasm of the parasite. They further showed that the trypanosomes are capable of absorption of the vitamin, since an increase in the ascorbic acid content of the blood of the host, brought about by injection, was rapidly followed by a corresponding increase in the quantity of the vitamin C in the parasites as demonstrated by staining with the specific vitamin C reagent.

The possibility that a similar absorption may take place in larger parasites has led us to investigate the localization of vitamin C in the ciliate Nyctotherus cordiformis Ehrenberg, the nematode Toxocara canis Werner (syn. Belascaris marginata), and in the trematode Opisthioglyphe ranae Frölich.

The nematodes were obtained from a post-mortem examination of the alimentary canal of a dog which had previously been fed on a normal mixed diet. The ciliates and trematodes were obtained from the alimentary canal of a freshly killed frog. The organisms, after removal from the host, were rinsed in saline to remove the intestinal debris, washed quickly in distilled water and fixed as described below. The nematodes were cut into small sections before fixation.

The basis of the histological demonstration of vitamin C is that under certain conditions, only this substance will bring about the reduction of silver nitrate to metallic silver. The technique was first introduced by Szent-Györgyi (1928), but the difficulty of introducing a fixing agent into the reagent was not solved until Giroud & Leblond (1934) produced a very specific reagent by using acetic acid combined with silver nitrate. Bourne (1936) has modified this reagent somewhat and it is his technique that is used in the present paper. This method is as follows :

The reagent consists of 5 % silver nitrate combined with a 5 % solution of acetic acid in the proportion of 5 c.c. of acid to each 100 c.c. of the silver solution. It is important to use the purest silver nitrate obtainable; B.D.H. ‘Analar’ salts were used in the present research. In the preparation and use of the reagent, absolute cleanliness in the preparation of the glassware is essential. All containers were first washed in soap and water, secondly with chromic acid, and finally rinsed with tap water, followed by glass-distilled water. Impregnation of the tissues was carried out in the dark to prevent possible decomposition of the silver salt by the action of light. The most satisfactory period was found to be about 20 hr. for the helminths, and 2–3 hr. for the ciliates. After impregnation, the unreduced silver was removed by placing the tissues in photographic ‘hypo’ for 2 hr. This was followed by several changes of distilled water— about an hour in each. The helminths were dehydrated, cleared, and embedded; sections were cut at 5μ. The ciliates were concentrated by centrifuging, and mounted whole by the albumen film method (Smyth, 1944).

Preparations were counterstained in 1 % orange G in absolute alcohol ; toning in gold chloride was also used to intensify the reduced silver in the tissues. A few preparations were treated with 5 % ammonia solution following a suggestion by Bourne, Barnett & Fisher (1941) that this increases the specificity of the method in some cases. It was found that with the present organisms, this treatment in no way altered the cytological picture as compared with that obtained in untreated preparations.

The fact that vitamin C is the only substance that will reduce silver nitrate in the presence of acetic acid has been proved by many workers, and it is not proposed to discuss the question here in any detail. Harris (1933) has shown that the tissues which give the highest figures for vitamin C content by titration also stain most intensively with silver nitrate ; he further demonstrated that in progressive scurvy, the decrease in ability of the tissues to reduce silver nitrate is compatible with the decrease in amount of vitamin C as estimated by titration. The silver nitrate-acetic acid reagent has been used by numerous workers especially on vertebrate glandular tissue, and there seems little doubt as to its specificity. Recently, some objections have been put forward by Barnett & Fisher (1943) from the results of staining mixtures of gelatin and ascorbic acid, and ground glass and ascorbic acid, but according to Bourne (1944) it is doubtful whether their results have any true significance.

Preparations of this ciliate showed that the vitamin C was distributed uniformly throughout the endoplasm in the form of spherules or disks of varying size (V, Fig. 1). These were never found in the ectoplasmic layer (E, Fig. 1) of the cytoplasm, nor were any present in the region of the mega-or micronucleus. In contrast with the uniform distribution of vitamin C in Nyctotherus, it is interesting to note that no trace of vitamin granules of any type were found in the cytoplasm of Opalina obtained from the same frog.

A brief note on the localization of vitamin C in this organism has already been published (Smyth & Hill, 1944). Fig. 2 shows a transverse section of Toxocara, and it can be seen that the vitamin is concentrated in considerable quantity in the cells of the gut. These cells, shown enlarged in Fig. 3, are laden with heavily impregnated granules (V) situated in the region between the nucleus and the free cell border, the densest region of impregnation being that just above the nucleus. A few granules are found just beneath the wall of the free border and a series of fine granules are present below the nucleus and lining the lower cell wall where it meets the external cuticle covering the gut (B, Fig. 3).

Compared with the intestinal cells, the amount of Vitamin distributed in the remaining tissues is exceedingly small. A few granules are found in each of the cells of the reproductive tubules (R, Fig. 2), but these in contrast with the granules of the gut cells are small and only visible under oil immersion. In the longitudinal muscles (M, Fig. 2) very fine diffuse grains are present. In all the other tissues— nerve cells, epidermis, etc.—only very few scattered grains are visible. In the section shown in Fig. 2, an elongated blackened bleb (K) can be seen in the body cavity. Since the food (F, Fig. 2) in the gut lumen shows some vitamin C present, it seems possible that this bleb represents a particle of food material which found its way into the body cavity when the nematode was cut into slices before fixation.

In this trematode, in contrast with Toxocara, the cells of the gut are completely lacking in granules of vitamin C. The distribution is largely confined to the thin walls of the excretory vessels. In Opisthioglyphe, the excretory system takes the form of two longitudinal canals which join to form a single median vessel in the posterior third of the body before passing to the posterior excretory pore. In Fig. 4 the section has been taken at the point of junction of the lateral excretory vessels. The walls of each of these is lined with impregnated granules of vitamin C, and the transverse connecting region (I, Fig. 4) shows an even heavier impregnation. In Fig. 5 the excretory canal is single but widened considerably preparatory to dividing into two. Here, too, the same impregnation is observed.

The vitamin is also seen in all sections as a very thin line of flattened granules lying beneath the epidermis (L, Fig. 4, 5). Throughout the remainder of the tissues very little vitamin is present. A few scattered granules are present in the parenchyma, longitudinal and diagonal muscle layers, and a small quantity is also localized in the tissues of the testes and the yolk glands.

The problem which now presents itself is how the presence of the vitamin in the tissues is to be interpreted. Since the parasites obtain their nourishment from the host, the most reasonable conclusion to be drawn is that they are able to absorb the vitamin from the food or tissues of the host. Indeed, if the host is fed on a normal mixed diet, we should expect such an absorption to take place. It is difficult to account for the absence of any vitamin granules in the astomatous Opalina, as compared with the presence of numerous granules in the cytoplasm of the stomatous Nyctotherus. The fact that this latter ciliate is a detritus feeder, whereas Opalina takes in nourishment over its whole body surface, may account for this difference.

In Toxocara, the fact that such relatively large concentrations of vitamin C are found in the gut cells is very strong evidence that absorption of vitamin takes place from the food of the host. Hirsch (1939) has shown that if isolated strips of intestine from a starved Ascaris are incubated in a 0·1% solution of vitamin C, the intestinal cells absorb the vitamin which becomes concentrated in the Golgi apparatus. In Toxocara, the presence of granules below the free border of the gut cells possibly represents the process of absorption actually taking place. It is interesting to note that in the turbellarian Dendrocoelum lacteum, a similar localization of the vitamin in the gut has been demonstrated by one of us (G.R.H., unpublished). Moreover, in specimens of Dendrocoelum placed in a weak solution of vitamin C (0·005 %), the number and size of the granules became increased appreciably, indicating that absorption of vitamin C via the gut cells could readily take place.

The results obtained in Opisthioglyphe are surprising in view of the distribution of the vitamin in Toxocara, and it is difficult to put forward any reasonable hypothesis to account for the almost exclusive localization of the vitamin to the excretory system. It was thought that the peculiar distribution might possibly be due to faulty technique or impure reagents, but repetition of the entire experiment using freshly prepared reagents and further specimens of Opisthioglyphe from different frogs, yielded identical results. It is difficult to see how the vitamin comes to be localized in the excretory canals if it is not absorbed by the cells of the gut in the first instance. Until further experimental work is carried out on this organism no definite conclusion can be reached.

The fact that parasites may be capable of absorption of vitamin C from the food or tissues of the host, may well lead to a revision of our theories of the host-parasite relationship. Many of the effects of parasites are due to the absorption of food material, especially in the case of nematodes. The great concentration of vitamin C in the gut cells of Toxocara seems to justify the conclusion that absorption of the vitamin has taken place. If a similar absorption takes place in human nematodes, as Hirsch’s results with isolated intestinal tissue of Ascaris seem to indicate, it is possible that in cases of very heavy infection the whole vitamin C metabolism will be thrown out of order. It is doubtful whether in a well-nourished individual sufficient quantities of the vitamin could be absorbed to produce harmful results. In an under-nourished organism, however, the vitamin C content may just be sufficient to prevent scorbutic conditions, and the introduction of nematodes in quantity may have the effect of sufficiently upsetting the vitamin C balance to induce scorbutic conditions.

As regards the ciliate studied, the same conclusions arrived at for the nematode may also be deduced. It is very doubtful whether intestinal ciliates are ever present in sufficient numbers to absorb an appreciable quantity of the vitamin. It is possible, of course, in the case of blood Protozoa—as was pointed out by Roskin & Nastiukova (1941)—that organisms such as trypanosomes may be present in sufficient quantity to produce an appreciable absorption.

Further experimental work will be needed especially on the effect of parasites on the metabolism of normal and scorbutic animals, before any generalized conclusion as to the part played by parasites in the vitamin C equilibrium of the host can be forthcoming. But it seems advisable that in considering the pathological effects of parasites— and especially nematodes—the possibility of vitamin absorption should not be overlooked.