A Study of the Mitochondria of Trypanosomes

—The following species of trypanosomes were available for study: Trypanosoma lewisi (Kent, 1880), Trypanosoma duttoni (Thirous, 1900), Trypanosoma melophagium (Flu, 1908), and Trypanosoma cruzi (Chagas, 1909), from the lewisi group, and Trypanosoma equiperdum (Doflem, 1901), from the evansi group (Hoare, 1936).

—For a study of the parasites in the vertebrate hosts films were made from the blood of experimentally infected rats, mice, and guinea-pigs. The subsequent treatment of the drawn films was based essentially on Minchin’s (1909) procedures. While still wet they were inverted immediately over, but not touching, a drop of 2 per cent, osmic acid solution in the bottom of a Petri dish. The slide remained, in the closed dish, suspended in osmic vapour until dry. It was then removed and dipped in 95 per cent, ethyl alcohol for several minutes, and set aside to dry. Such films were stained in a number of ways, that is, with Giemsa, Wright’s, and Altmann’s acid fuchsin differentiated with aurantia. Wet films were also fixed by flooding with Champy’s fluid (3 per cent, potassium bichromate, 7 parts ; 1 per cent, chromic acid, 7 parts ; and 2 per cent, osmic acid, 4 parts) and subsequently mordanted and stained as Champy-Kull preparations (Lee, 1937). All slides were mounted with a cover-glass, using neutral cedar oil as a medium. The best results for the demonstration of mitochondria were obtained following osmic acid-vapour fixation.

Lipoid inclusions were tested for with Sudan IV; such preparations were mounted in glychrogel (Zwemer, 1933; Wotton and Zwemer, 1935; Wotton, 1936).

The bodies of the arthropod vector of Trypanosoma melophagium, Melophagus ovinus, after suitable fixation, were dehydrated and carried through the customary paraffin imbedding technique. Some specimens were fixed in warm Bouin (B-3 mixture at 37·0° C.), then stained with haematoxylin and eosin, while others were immersed in Champy’s fluid and subsequently stained with Altmann’s acid fuchsin.

Material from cultures of Trypanosoma cruzi was placed on a clean slide with a sterile pipette, and inverted over a drop of osmic acid solution and manipulated thenceforth in every way similar to the drawn films.

The mitochondria of all species of trypanosomes appear as rather slender rod-shaped bodies in the cytoplasm, staining specifically with Altmann’s acid fuchsin. They are generally few in number, seldom more than twelve being observed in any one cell. In the adult form, they are generally arranged parallel to the long axis of the cell, their alignment being accounted for merely by the slenderness of the parasite. In division forms variations from this arrangement were observed.

Trypanosoma lewisi (figs. 1-8, Pl. 10).—The characteristic distribution of the mitochondria as seen in the adult form of Trypanosoma lewisi is subject to certain variations in reproduction forms. They often tend to collect in the end of the cell opposite the nucleus and frequently are situated at a right angle to the long axis of the cell-body (figs. 3, 4, and 5, Pl. 10). Other alterations in the orientation such as clumping around the nucleus, as reported by Shipley (1916), occur. During cell division the mitochondria assume a passive role and are distributed at random among the daughter cells (figs. 3 and 4, Pl. 10).

Highly refractile granules are often observed in close apposition to the mitochondria in the acid fuchsin stained slides. These same granules, varying in size, were tinctured by typical fat dyes, and blackened by osmication in other preparations. Such cyto-chemical reactions strongly indicate the fatty nature of these granules.

These fatty granules are seen chiefly among the mitochondria situated in the posterior end of the cell, a condition especially prominent in the growth forms of the trypanosome, where the mitochondria reach their maximum size and number. We are unable to say whether the chondriosomes exert directly a synthesizing action upon these granules or merely provide a large interface upon which such reactions can take place.

Films treated by the Kolatchew method used by cytologists for demonstrating the Golgi apparatus reveal the osmiophilic nature of the parabasal body of the trypanosome (Duboscq and Grassé, 1933). Surrounding the parabasal body and blepharoplast (figs. 2, 3, and 5-8, Pl. 10) is a clear zone which may assume the size and shape of a very large vacuole. It is clearly brought out by counter staining osmicated specimens of Trypanosoma lewisi with crystal violet. If the parabasal body of the trypanosome cell is the analogue of the Golgi apparatus, as has been postulated by some cytologists, then the clear zone surrounding it may be considered as some form of secretion product.

—So far as the mitochondrial arrangement is concerned Trypanosoma dutf oni shows no essential difference from Trypanosoma lewisi. Trypanosoma duttoni has been classified by most authorities as a variation of the lewisi species (Wenyon, 1926).

Trypanosoma melophagium (figs. 17 and 18, Pl. 10).— Try panoso ma melophagium was obtained from the alimentary tract of the sheep ked, Melophagus ovinus. In the arthropod vector it exists chiefly in the crithidial or the motacyclic form, although it returns to the true trypanosome form in the blood-stream of the sheep. In the crithidial stage in the ked, no difference was noted in the mitochondrial mor-pliology from that observed in the trypanosome form of other species here studied.

—In this species the mitochondria are somewhat fewer in number, though in other respects there is no great variation in the cytology of either the animal or cultural (crithidial) forms of Trypanosoma cruzi from the other trypanosomes observed in the lewisi group.

Trypanosoma equiperdum (figs. 8, 10, and 11, Pl. 10).—Although this trypanosome belongs to the e van si group and is remote taxonomically from the trypanosomes just discussed of the lewisi group, a study of its cellular component discloses that it resembles the other species observed above.

Considering the morphological similarity between the mitochondria of trypanosomes and the mitochondria found in higher spedea of animate, there seems great likelihood of a similarity in function. Though studies of these cytoplasmic elements among cells of the higher forms have been numerous, their specific nature and function remains obscure. It is evident from the recent work of Hirsch (1932 a and V) and Duthie (1933), however, that mitochondria are, along with the Golgi apparatus (Bowen, 1926 and 1929), related to the synthesis of secretion products in glandular cells of higher forms, their orientation in the cell being associated with their metabolism (Pollister, 1932). A similar metabolic activity may be implied for the mitochondria in trypanosomes. From our studies on Trypanosoma lewisi it is suggested that this function is connected with fat metabolism.

Mitochondria have been demonstrated in the adult forms of the following species of trypanosomes: Trypanosoma lewisi, Trypanosoma duttoni, Trypanosoma cruzi, and Trypanosoma equiperdum. They have been shown also in the crithidial forms of Trypanosoma cruzi from culture, and of Trypanosoma melophagium from the insect vector, Melophagus ovinus. No essential morphological variations in the mitochondria among the five species of trypanosomes studied, nor in those among the several growth stages, were observed.

All the figures in the plate were outlined with a camera lucida, and were drawn at a magnification of 3,000 diameters, reduced to 2,000 diameters in production. Figs. 1-9 were drawn from preparations fixed in osmic vapour and stained with Altmann’s acid fuchsin, or osmicated by the Kolatchew method and counter-stained with crystal violet ; figs. 10-18 were drawn from preparations fixed in Champy’s fluid and stained with Altmann’s acid fuchsin.

Figs. 1-4.—Show mitochondria and refractile granules in Trypanosoma lewisi. Fig. 1 shows the adult form of the trypanosome, while figs. 3-4 illustrate various division forms.

Figs. 5-8.—Show the osmiophilic parabasal body in Trypanosoma lewisi.

Fig. 9.—T rypanosomaduttoni with mitochondria in its cytoplasm.

Figs. 10-12.-—Mitochondria in Trypanosoma equiperdum. Figs. 10 and 11 are division forms.

Fig. 13.—Monocyte with mitochondria, from same slide as figs. 14 and 15.

Figs. 14-15.—Trypanosoma cruzi, from vertebrate host, showing mitochondria.

Fig. 16.—A crithidial form of Trypanosoma cruzi from a culture showing mitochondria.

Figs. 17-18.—Crithidial forms of Trypanosoma melophagium attached to the alimentary tract of the sheep ked (Melophagus ovinis).

K., blepharoplast; p., parabasal body; F., flagellum; M., mitochondria; G., refractile granule; N., nucleus; c., columnar cell of Melophagus ovinus.