THE DISTRIBUTION OF VITAMIN C IN NYCTOTHERUS CORDI-FORMIS EHRENBERG, OPISTHIOGLYPHE RANAE FROLICH, AND

TOXOCARA CANIS WERNER

BY J. D. SMYTH, W. J. BINGLEY AND G. R. HILL, Department of Zoology,University College, Leicester

{Received 23 March 1944)

(With Five Text-figures)

INTRODUCTIONThe possibility that internal parasites may play apart in the vitamin metabolism of the host was firstsuggested by Roskin & Nastiukova (1941). Workingon Trypanosoma brucei in guinea-pigs, these workersdemonstrated the presence of vitamin C granules inthe cytoplasm of the parasite. They further showedthat the trypanosomes are capable of absorption ofthe vitamin, since an increase in the as'corbic acidcontent of the blood of the host, brought about byinjection, was rapidly followed by a correspondingincrease in the quantity of the vitamin C in theparasites as demonstrated by staining with thespecific vitamin C reagent.

The possibility that a similar absorption may takeplace in larger parasites has led us to investigate thelocalization of vitamin C in the ciliate Nyctotheruscordiformis Ehrenberg, the nematode Toxocara cantsWerner (syn. Belascaris marginatd), and in thetrematode Opisthioglyphe ranae Frolich.

MATERIAL AND METHODSThe nematodes were obtained from a post-mortemexamination of the alimentary canal of a dog whichhad previously been fed on a normal mixed diet. Theciliates and trematodes were obtained from thealimentary canal of a freshly killed frog. Theorganisms, after removal from the host, were rinsedin saline to remove the intestinal debris, washedquickly in distilled water and fixed as describedbelow. The nematodes were cut into small sectionsbefore fixation.

The basis of the histological demonstration ofvitamin C is that under certain conditions, only thissubstance will bring about the reduction of silvernitrate to metallic silver. The technique was firstintroduced by Szent-Gyorgyi (1928), but the diffi-culty of introducing a fixing agent into the reagentwas not solved until Giroud & Leblond (1934) pro-duced a very specific reagent by using acetic acidcombined with silver nitrate. Bourne (1936) hasmodified this reagent somewhat and it is his tech-nique that is used in the present paper. This methodis as follows:

The reagent consists of 5 % silver nitrate com-bined with a s % solution of acetic acid in theproportion of 5 c.c. of acid to each 100 c.c. of thesilver solution. It is important to use the purestsilver nitrate obtainable; B.D.H. ' Analar' salts wereused in the present research. In the preparation anduse of the reagent, absolute cleanliness in the pre-paration of the glassware is essential. All containerswere first washed in soap and water, secondly withchromic acid, and finally rinsed with tap water,followed by glass-distilled water. Impregnation ofthe tissues was carried out in the dark to preventpossible decomposition of the silver salt by theaction of light. The most satisfactory period wasfound to be about 20 hr. for the helminths, and2-3 hr. for the ciliates. After impregnation, theunreduced silver was removed by placing thetissues in photographic 'hypo' for 2 hr. This wasfollowed by several changes of distilled water—about an hour in each. The helminths were de-hydrated, cleared, and embedded; sections were cutat 5/u.. The ciliates were concentrated by centri-fuging, and mounted whole by the albumen filmmethod (Smyth, 1944).

Preparations were counterstained in 1 % orange Gin absolute alcohol; toning in gold chloride was alsoused to intensify the reduced silver in the tissues.A few preparations were treated with 5 % ammoniasolution following a suggestion by Bourne, Barnett& Fisher (1941) that this increases the specificity ofthe method in some cases. It was found that withthe present organisms, this treatment in no wayaltered the cytological picture as compared with thatobtained in untreated preparations.

VALIDITY OF THE VITAMIN CREACTION

The fact that vitamin C is the only substance thatwill reduce silver nitrate in the presence of aceticacid has been proved by many workers, and it isnot proposed to discuss the question here in anydetail. Harris (1933) has shown that the tissueswhich give the highest figures for vitamin C contentby titration also stain most intensively with silver

J. D. SMYTH, W. J. BINGLEY and G. R. H I L L

nitrate; he further demonstrated that in progressivescurvy, the decrease in ability of the tissues toreduce silver nitrate is compatible with the decreasein amount of vitamin C as estimated by titration.The silver nitrate-acetic acid reagent has been usedby numerous workers especially on vertebrateglandular tissue, and there seems little doubt as toits specificity. Recently, some objections have beenput forward by Barnett & Fisher (1943) from theresults of staining mixtures of gelatin and ascorbicacid, and ground glass and ascorbic acid, butaccording to Bourne (1944) it is doubtful whethertheir results have any true significance.

OBSERVATIONSNyctotherus. Preparations of this ciliate showed

that the vitamin C was distributed uniformlythroughout the endoplasm in the form of spherulesor disks of varying size (V, Fig. 1). These werenever found in the ectoplasmic layer (E, Fig. 1) of

•=—MN

Fig. 1. Portion of Nyctotherus stained by the silvernitrate-acetic acid technique. Camera lucida drawing.E, ectoplasm; MN, meganucleus; V, vitamin C granules.

the cytoplasm, nor were any present in the regionof the mega- or micronucleus. In contrast with theuniform distribution of vitamin C in Nyctotherus, itis interesting to note that no trace of vitamingranules of any type were found in the cytoplasm ofOpalina obtained from the same frog.

Toxocara. A brief note on the localization ofvitamin C in this organism has already been pub-lished (Smyth & Hill, 1944). Fig. 2 shows a trans-verse section of Toxocara, and it can be seen thatthe vitamin is concentrated in considerable quantityin the cells of the gut. These cells, shown enlargedin Fig. 3, are laden with heavily impregnated granules(V) situated in the region between the nucleus andthe free cell border, the densest region of impregna-tion being that just above the nucleus. A few gran-ules are found just beneath the wall of the free borderand a series of fine granules are present below the

nucleus and lining the lower cell wall where itthe external cuticle covering the gut (B, Fig.

Compared with the intestinal cells, the amount ofVitamin distributed in the remaining tissues is ex-ceedingly small. A few granules are found in eachof the cells of the reproductive tubules (R, Fig. 2),but these in contrast with the granules of the gutcells are small and only visible under oil immersion.In the longitudinal muscles {M, Fig. 2) very finediffuse grains are present. In all the other tissues—nerve cells, epidermis, etc.—only very few scatteredgrains are visible. In the section shown in Fig. 2,an elongated blackened bleb (K) can be seen in thebody cavity. Since the food (F, Fig. 2) in the gutlumen shows some vitamin C present, it seems pos-sible that this bleb represents a particle of food'material which found its way into the body cavitywhen the nematode was cut into slices beforefixation.

A'

Fig. 2. Transverse section of Toxocara with externalcuticle removed. Camera lucida drawing. Silver nitrate-acetic acid preparation. E, excretory canal; F, food ingut; G, gut cells laden with vitamin C granules; K,artifact; M, longitudinal muscles; R, reproductive tubes.

Opisthioglyphe. In this trematode, in contrast withToxocara, the cells of the gut are completely lackingin granules of vitamin C. The distribution is largelyconfined to the thin walls of the excretory vessels.In Opisthioglyphe, the excretory system takes theform of two longitudinal canals which join to form asingle median vessel in the posterior third of thebody before passing to the posterior excretory pore.In Fig. 4 the section has been taken at the point ofjunction of the lateral excretory vessels. The walls ofeach of these is lined with impregnated granules ofvitamin C, and the transverse connecting region(/, Fig. 4) shows an even heavier impregnation. InFig. 5 the excretory canal is single but widened con-siderably preparatory to dividing into two. Here, too,the same impregnation is observed.

The distribution of vitamin C

#e vitamin is also seen in all sections as a veryline of flattened granules lying beneath the

epidermis (L, Fig. 4, 5). Throughout the remainderof the tissues very little vitamin is present. A fewscattered granules are present in the parenchyma,longitudinal and diagonal muscle layers, and a smallquantity is also localized in the tissues of the testesand the yolk glands.

Fig. 3. Intestinal cells of Toxocara. Silver nitrate-aceticacid preparation. Camera lucida drawing. B, cuticularcovering of gut; N, nucleus; V, vitamin C granules.

DISCUSSIONThe problem which now presents itself is how thepresence of the vitamin in the tissues is to be inter-preted. Since the parasites obtain their nourishmentfrom the host, the most reasonable conclusion to bedrawn is that they are able to absorb the vitamin fromthe food or tissues of the host. Indeed, if the host isfed on a normal mixed diet, we should expect suchan absorption to take place. It is difficult to accountfor the absence of any vitamin granules in theastomatous Opalina, as compared with the presenceof numerous granules in the cytoplasm of thestomatous Nyctotherus. The fact that this latterciliate is a detritus feeder, whereas Opalina takes innourishment over its whole body surface, mayaccount for this difference.

In Toxocara, the fact that such relatively largeconcentrations of vitamin C are found in the gutcells is very strong evidence that absorption ofvitamin takes place from the food of the host. Hirsch

(1939) has shown that if isolated strips of intestinefrom a starved Ascaris are incubated in a o-i %solution of vitamin C, the intestinal cells absorb thevitamin which becomes concentrated in the Golgiapparatus. In Toxocara, the presence of granulesbelow the free border of the gut cells possibly repre-sents the process of absorption actually taking place.It is interesting to note that in the turbellarian

Fig. 4. Transverse section of Opisthioglyphe showingjunction of lateral excretory canals. Silver nitrate-aceticacid preparation. A, lateral excretory vessels; G, gut;/, junction of lateral canals heavily laden with vitamin C;L, layer of vitamin C grains beneath epidermis; S, eggsin uterus; Y, yolk glands.

Fig. 5. Transverse section of Opisthioglyphe showingmedian excretory vessel prior to division. Camera lucidadrawing. Silver nitrate-acetic acid preparation. A,median excretory vessel laden with vitamin C granules;G, gut; L, layer of vitamin C granules beneath epidermis;S, eggs; Y, yolk glands.

Dendrocoelum lacteum, a similar .localization of thevitamin in the gut has been demonstrated by one ofus (G.R.H., unpublished). Moreover, in specimensof Dendrocoelum placed in a weak solution ofvitamin C (0-005 %)> t n e number and size of thegranules became increased appreciably, indicatingthat absorption of vitamin C via the gut cells couldreadily take place.

The results obtained in Opisthioglyphe are sur-prising in view of the distribution of the vitamin in

i6 J. D. SMYTH, W. J. BINGLEY and G. R. H I L L

Toxocara, and it is difficult to put forward anyreasonable hypothesis to account for the almostexclusive localization of the vitamin to the excretorysystem. It was thought that the peculiar distributionmight possibly be due to faulty technique or impurereagents, but repetition of the entire experimentusing freshly prepared reagents and further speci-mens of Opisthioglyphe from different frogs, yieldedidentical results. It is difficult to see how thevitamin comes to be localized in the excretory canalsif it is not absorbed by the cells of the gut in thefirst instance. Until further experimental work iscarried out on this organism no definite conclusioncan be reached.

The fact that parasites may be capable of absorp-tion of vitamin C from the food or tissues of the host,may well lead to a revision of our theories of thehost-parasite relationship. Many of the effects ofparasites are due to the absorption of food material,especially in the case of nematodes. The greatconcentration of vitamin C in the gut cells of Toxocaraseems to justify the conclusion that absorption of thevitamin has taken place. If a similar absorption takesplace in human nematodes, as Hirsch's results withisolated intestinal tissue of Ascaris seem to indicate,it is possible that in cases of very heavy infection thewhole vitamin C metabolism will be thrown out oforder. It is doubtful whether in a well-nourishedindividual sufficient quantities of the vitamin couldbe absorbed to produce harmful results. In anunder-nourished organism, however, the vitamin Ccontent may just be sufficient to prevent scorbuticconditions, and the introduction of nematodes inquantity may have the effect of sufficiently upsettingthe vitamin C balance to induce scorbutic conditions.

As regards the ciliate studied, the same conclusionsarrived at for the nematode may also be deduced.It is very doubtful whether intestinal ciliates are everpresent in sufficient numbers to absorb an appreci-able quantity of the vitamin. It is possible, of course,in the case of blood Protozoa—as was pointed outby Roskin & Nastiukova (1941)—that organismssuch as trypanosomes may be present in sufficientquantity to produce an appreciable absorption.

Further experimental work will beespecially on the effect of parasites on thebolism of normal and scorbutic animals, before anygeneralized conclusion as to the part played byparasites in the vitamin C equilibrium of the hostcan be forthcoming. But it seems advisable that inconsidering the pathological effects of parasites—and especially nematodes—the possibility of vitaminabsorption should not be overlooked.

SUMMARY1. The localization of vitamin C in Nyctotherus

cordiformis, Toxocara cards and Opisthioglyphe ranaewas investigated by means of the silver nitrate-acetic acid technique.

2. In Nyctotherus, the vitamin C is distributeduniformly throughout the cytoplasm in the form ofglobules of varying sizes. No vitamin is present inthe ectoplasmic layer or in the region of the nuclei.In the same preparations specimens of Opalina showno trace of any vitamin C present.

3. In Toxocara, the vitamin is localized in rela-tively large quantities in all the cells of the intestine.In these cells it is aggregated mainly in the regionbetween the nucleus and the free cell border. Aseries of fine granules of the vitamin also line thelower cell walls. Only a few scattered granules arepresent in the remaining tissues.

4. In Opisthioglyphe, the vitamin is concentratedon the walls of the excretory system which in thistrematode has the form of two longitudinal canalsjoining in the posterior region to form a singlemedian canal. A line, of disk-like elements of thevitamin is also present immediately below theepidermis. In the remaining tissues only very smallscattered grains are found.

5. It is believed that in the case of Toxocara, thepresence of the vitamin in the gut cells may indicatethat the parasite is capable of absorption of vitaminC from the food of the host.

6. It is suggested that in heavy parasitic infectionsthe parasites may play some part in the disturbanceof the vitamin C balance of the host.

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HIRSCH, G. C. (1939). Form und Stoffwechsel der Golgi-Korper. Protoplasma Monographs. Berlin.

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