Electron microscope studies of motile stages of malaria parasites

27 TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE. Vol. 57. No. 1. January, 1963.

COMMUNICATIONS

ELECTRON IV.

MICROSCOPE

S T U D I E S OF M O T I L E PARASITES

STAGES

OF MALARIA

THE FINE STRUCTURE OF THE SPOROZOITES OF FOUR SPECIES OF PLASMODIUM BY P. C. C. G A R N H A M , R. G. BIRD AND J. R. BAKER*

London School of Hygiene and Tropical Medicine

The first and second papers of this series (GARNHAM et al., 1960, 1961) were concerned with the sporozoites of Haemamoeba and Laverania, respectively, and described certain details of their morphology as revealed by electron microscopy. Some structural differences were detected between the two genera, and it was thought at first that the possession of a micropyle by L. falcipara and its apparent absence in H. gallinacea constituted an important distinction : however, when further specimens of the latter parasite were examined, a typical micropyle was found. We did not expect to discover significant differences in the appearance of Plasmodium sporozoites, but we felt that a variety of species of this genus should be examined and their fine structure compared with our earlier findings. MATERIAL AND METHODS Salivary glands of Anopheles stephensi infected with Plasmodium vivax or P. bastianeUii, of A maculipennis atroparvus infected with P. bastianellii, of A. aztecus infected with P. brasilianum and of A. garnbiae infected with P. ovale were dissected when sporozoites were present and fixed in ice-cold 1 per cent. osmium tetroxide solution kept at p H 7 or 7.3 with Michaelis' (1931) buffer. Fixation time varied between ¼ and 2 hours, but was usually about 1½ hours. Subsequent treatment, including staining with tungstophosphoric acid for ½ hr., followed the procedure given in the first paper of this series (GARNHAM et al., 1960). OBSERVATIONS

Plasmodium vivax. In dried preparations on slides, the sporozoite of P. vivax is seen as a thin body about 1~ in width and 11.5 ~ in length ; the ends are pointed or one may be slightly rounded. In such preparations the nucleus is very often divided into two or more portions, an appearance not seen in our material as prepared for electron microscope examination, in which the greatest single length of any sporozoite in section was 10.5 ~. With the electron microscope, the sporozoite is seen enclosed in a thick pellicle which, except anteriorly, appears at low magnification as two dense layers separated by a wider and lighter core. The whole pellicle measures about 30 m~z in width. At higher magnification the outer dense layer looks like a " unit membrane," about 7.5 m~ thick, as described by *We acknowledge with much pleasure the provision of mosquitoes infected with P. vivax by Mr. P. G. Shute, and with P. ovale by Dr. R. S. Bray; and the technical assistance of Mr. P. Healey.

28

F I N E STRUCTURE OF THE SPOROZOITES OF F O U R SPECIES OF

Plasmodium

ROBERTSON (1960). Sometimes there is a suggestion of periodicity in the pellicle, i.e., regular breaks in continuity or vertical ridges in the core, but this appearance may be an artefact. The posterior end is blunt and U-shaped, while an apical cup occupies the rather flattened anterior end. The latter is made up of three or four concentric rings apparently linked to the peripheral fibrils (Fig. 2). The pellicle is broken at one point near, and almost invariably anterior to, the nucleus by the micropyle. At this point, the pellicle dips at right angles into the interior, its two dense layers thickening to form a stout wall to the micropyle, which is sealed, if at all, by only a very thin membrane (Fig. 5). The width of the pit so formed is 60 m~z, and its depth 100 m~z. In surface view the micropyle has a smooth oval or circular contour (Figs 1, 4). Inside the pellicle run the hollow peripheral fibrils, arranged asymmetrically around the circumference of the sporozoite, and possibly extending from one extremity to the other. Each fibril is 15-20 m~z in diameter. There appear to be 10 fibrils distributed around more than half the circumference, and a single one isolated in the middle of the other half (Fig. 6). The paired organelle (Figs 1, 3) is made up of two narrow lobes of fairly uniform width (40-60 mtz) which curve back towards the nucleus from the apical cup, in which they are believed to open. One lobe may be split into two diverticula. In addition there is a large number of equally dense structures which we now think to be sections of highly convoluted tubules, about 40 mtz thick, possibly equivalent to LUDVfK'S (1958) " sarconemes" (Figs 1, 4, 5) ; they are more numerous in front of the nucleus, but exist also posteriorly. These convoluted tubules are absent from the nucleus region, and it is difficult to believe that they are continuous from one end of the sporozoite to the other. The body of the sporozoite is so filled with convoluted tubules that mitochondria may be undetectable, and anyhow they are present in only small numbers. In longitudinal section they are linear, rather narrow and contain circles which may be villi cut in cross section. Occasionally a " fenestrated button " of well defined outline may be visible (Fig. 6a) : these structures are up to 60 m~z in diameter. The nucleus has a granular texture and measures up to 2.3 x 0.48 ~z ; it almost fills the whole width of the sporozoite (Fig. 4). The anterior end is drawn out into a snout, often directed towards the micropyle. No clear nuclear membrane has been detected. Plasmodium bastianellii. The living sporozoite of this species measures 15 ~z in length ; in the present study, longitudinal sections of up to 9.4 ~z in length only have been seen.

LEGENDS FOR FIGURES All figures are electron micrographs of thin sections of Plasmodium sporozoites in mosquito salivary glands. List of magnifications :

Figs 1-4 x 48,000 Fig. 13 x 80,000 Fig. 5 x 40,000 Figs 14 and 15 x 48,000 Fig. 6 x 48,000 Fig. 16 x 30,000 Fig. 6a x 240,000 Fig. 17 x 80,000 Figs 7 and 8 x 16,000 Fig. 18 x 48,000 Figs 9 and 10 x 48,000 Fig. 19 x 12,000 Fig. 11 x 39,000 Figs 20 and 21 x 48,000 Fig. 12 x 80,000 Figs 22 and 23 x 30,000 Fig. 12a x 100,000 Abbreviations : AC--apical cup; B--"fenestrated button"; C--convoluted tubules; CR-concentric rings around apical cup; F--peripheral fibrils; GCN--salivary gland cell nucleus; M--mitochondrion ; MP--micropyle ; N--nucleus ; P--pellicle ; PO--paired organelle.

PLATE

FIGS

I-6a.

P. vizmx

(To face page 28)

: longitudinal

sections

and

I

a transverse sporozoites.

section

(Fig.

6) through

different

regions

of

PLATE

FIGS 7-13.

P. bastianellii

through

same

the

sporozoite.

: longitudinal “unit

Fig. 12a membrane”

and transverse (Figs is part of a transverse structure of both

II

9, 12, 13) sections. Figs 7 and section at high magnification, layers of the pellicle.

8 are sections showing the

PLATE III

FIGS 14-19. Fig.

P. ovale : longitudinal

19 (Plate IV) is a low-power lobe, showing longitudinal

(Figs

14, 16), oblique

(Fig. 15) and transverse

micrograph of a section through part and transverse sections of sporozoites

(Figs 17, 18) sections.

of one infected salivary gland within the gland cells.

22 FIGS

20-23.

P.

oblique brusiZianum

:

longitudinal

(Figs

20,

23),

(Fig.

22) and

transverse

(Fig.

21)

sections.

P . C. C. G A R N H A M , R. G. B I R D A N D J . R. BAKER

29

The pellicle of this species is about 25 m~ thick and, at lower magnifications (Fig. 9), appears as two electron-dense layers separated by a zone of less dense material. At higher magnification (Fig. 12a) each of the dense layers may be resolved further to resemble the unit membrane (ROBERTSON, 1960). The intermediate zone shows variations in texture which make the whole peUicle look corrugated. The anterior end is formed by a flattening in which lie several concentric rings. Near the nucleus is the prominent micropyle of exactly similar structure to that of a P. vivax sporozoite (Figs 9, 10). There is never more than one micropyle. The peripheral fibrils run posteriorly from the apical cup immediately beneath the pellicle. They are hollow and are arranged asymmetrically--10 occupying about two-thirds of the circumference, and a single one by itself (Figs 12, 13). The paired organelle (Figs 7, 8) consists of two rather thin lobes (50-60 m~ wide) which extend at least to the nucleus. There are also the convoluted tubules, in these preparations not so numerous as in P. vivax and presumably less convoluted because in section they are more often linear than round or oval. Mitochondria are more often visible in this species (Fig. 11) ; their appearance is similar to those seen in P. vivax and they measure up to 1 in length. The nucleus in this material has a clearly defined membrane (Fig. 11) and the usual truncated extremity directed towards the micropyle (Fig. 10). The nucleus in cross section often shows smaller or larger depressions on one side, suggestive of some irregularity of its surface which is invisible however in longitudinal sections, unless it is part of the " snout." Plasmodium ovale. In the struggle to confirm the specific validity of this organism, JAMES et al. (1933) laid great stress on the difference between the sporozoites of P. ovale and P. vivax, and stated that no one who examined preparations side by side could doubt that they belonged to different species. We therefore included P. ovale in our present observations, hoping to substantiate these findings, although w e (GARNHAM et al., 1955) had already shown that the sporozoites were of a normal length - - 12 ~ in dried preparations --instead of being excessively short as stated by James. The thick pellicle (about 25 m~) consists of the usual three layers, the inner and outermost being of equal width, and with periodic zones of greater and lesser electron density. The pellicle is interrupted at the apical cup, and at the micropyle (Figs 14, 17), a structure of the same degree of prominence as in other species. The walls of the micropyle, which here again is found anterior to the nucleus, are much thickened and there appears to be an intact membrane at the base of the depression. Its internal dimensions are as follows : depth 70 m~, width up to 50 m~.. The peripheral fibrils are hollow and have the typical asymmetrical distribution beneath the pellicle : there appear to be 12 arranged around two-thirds of the circumference, and an isolated one in the remaining third (Fig. 17). The paired organelle shows certain features which are i n t e r e s t i n g - if not artefacts of fixation. Two lobes extend backwards from the apex ; after a short course the lobes may suddenly expand into a wider body, and both this and the more anterior part are vesiclelike, i.e., their central region is less dense than the enclosing walls, suggesting a secretory function. Fairly numerous convoluted tubules, cut in cross section, are present in the interior, together with the occasional but well-defined mitochondria (Fig. 15), which resemble those

30

FINE STRUCTURE OF THE SPOROZOITES OF FOUR SPECIES OF

Plasmodium

of the species described above. The nucleoplasm (Figs 14, 26) is uniformly granular and is enclosed by a membrane. Plasmodium brasilianum. This quartan parasite of New World monkeys develops poorly in the usual species of Anopheles maintained in laboratories, and the sporogonic stages are difficult to elicit. Our material therefore was neither abundant nor strongly viable ; in consequence, the following description lacks precision. The pellicle measures about 30 m~ in width and again appears as two electron-dense layers separated by a less dense region. Although the apical cup has not been seen, a flattening towards the anterior extremity can be detected (Fig. 23). A well-defined micropyle is visible in some sections (Fig. 20), and presents exactly the same appearance as those of the other species. Peripheral fibrils exist (Fig. 21) ; they are hollow, but their exact number has not been determined. The " paired organelle " i s an obvious structure and shows some of the features exhibited by the sporozoite of P. ovale ; thus its lobes tend to become dilated posteriorly, its whole appearance suggesting a secretory function. In one section (Fig. 20) the " paired " organelle showed triple lobes but unfortunately we cannot be sure that this is a constant feature. Many convoluted tubules were seen in some sections, but no satisfactory picture of the nucleus or mitochondria has been obtained. DISCUSSION

Sporozoites of six species of malaria parasites have now been studied by electron microscopy following fixation with osmium tetroxide. A comparison of their fine structures shows that only minute differences exist between them, especially in the case of the mammalian forms : the Table summarizes these apparent differences. We are by no means certain that the small differences noted among the species of Plasmodium and Laverania are to be regarded as specific characteristics. For instance, it has not been finally demonstrated that the number of peripheral fibrils is constant among the individuals of a species. Again, the three-layered structure of the inner and outer layers of the pellicle, visible particularly in our preparations of P. bastianellii and--to a lesser extent--P, vivax, is probably present in all the species though not demonstrated by our earlier work. The pellicle of Lankesterella garnhami has a similar s t r u c t u r e ( G A R N H A M et al., 1962b) although its component parts appear thinner. Having now examined a wider variety of species, we are able to revise some of the conclusions that we put forward in the earlier papers with reference to the " convoluted tubules." These organelles we first called mitochondria, and later, very tentatively, lysosomes. The true mitochondrion is a much larger structure with a definite limiting membrane and villi, and is well seen in some of the Plasmodium species ; the numerous small round, oval or elongated bodies are now thought to be homologous with LUDViK'S (1958) " sarconemes " - - a n inappropriate word because they are no more confined to Sarcoc3~stis than are the " toxonemes " (GUSTAVSONet al., 1954 ; LUDViK, 1956) to Toxoplasma ; we therefore propose the general term " convoluted tubule " to replace both " sarconeme " and " toxoneme " • it is probable that Gustavson and Ludvlk included in the latter term both the convoluted tubules and the paired organelle. With regard to the structures we have called " fenestrated buttons" in this and previous papers (GARNHAMet al., 1960, 1961, 1962c), it is interesting to note that a similar appearance has been seen in the electron microscope when a small droplet of 1 per cent. tungstophosphoric acid s o l u t i o n - having been evaported on to a carbon f i l m - has been exposed to

P. C. C. GARNHAM~ R. G. BIRD AND ]'. R. BAKER

31

an electron beam of high intensity. We think, therefore, that although these structures indicate a cell constituent with strong affinity for tungstophosphoric acid, their fenestrated appearance is an artefact. Perhaps the most interesting structure in the sporozoite is the micropyle, not only a constant feature of all these sporozoites, but present in the sporozoan parasite L. garnhami (GARNHAM et al., 1962b), and in Toxoplasma (GARNHAM et al., 1962a) ; LUDViK (1962) recently has demonstrated it in the sporozoites of H. cathemeria, in the so-called " M " organism and in Sarcocvstis, pointing out in regard to the last parasite that the micropyle or " lateral pore " is visible by light microscopy in a constant position in the spore. We think that the possession of a micropyle indicates that the organism belongs to the Sporozoa. Our suggestion (1961) that the micropyle is the place of emergence of a " sporoplasm " to initiate exo-erythrocytic schizogony still appears valid, though our attempts to prove the hypothesis by inoculating enormous numbers of sporozoites into an animal, and identifying the " empty husk " of the sporozoite within a liver parenchyma cell, have so far been unsuccessful. It is easy to visualize a sporozoite of such structure lying dormant in the tissues to give rise at a later date to a delayed primary infection, or relapse, as was suggested by SHUTE (1946) and others ; the sporoplasm remaining inactive inside the wall, with the micropyle sealed. Prolonged survival of the emerged sporoplasm in an ageing parenchyma cell of the liver would appear to be impossible, as the latter dies after some months. TABLE (Summary). Peripheral fibrils

Species

Paired organelle

H. gallinacea L. falcipara

11 + 1 14+1

Large and bulbous Narrow and sinuous

P. vivax

10 + 1

Narrow

P. bastianellii

10+1

Narrow

P. ovale

12+1

Narrow with posterior bulb ; "vesicular" With bulb ; ?triple

?

P. brasilianum

Convoluted tubules Scanty Numerous ; less curved (i.e., linear in section) Numerous ; much curved (i.e., circular in section) Less numerous : less curved (i.e., linear in section) Fairly numerous Fairly numerous

(Constant features, such as nucleus, mitochondria, micropyle and thick pellicle have been omitted). REFERENCES GARNHAM,P. C. C., BAKER,J. R. & BIRD, R. G. (1962a). Brit. reed. J., 1, 83. (1962b). J. Protozool., 9, 107. ---, BIRD, R. G. & BAKER,J. R. (1960). Trans. R. Soc. trop. Med. Hyg., 54, 274. --, - &- (1962c). Ibid., 56, 116. & BRAY,R. S. (1961). Ibid., 55, 98. ---, BRAY,t/. S., COOPER,W., LAINSON,R., AWAD, F. I. & WILLIAMSON,J. (1955). Ibid., 49, 158. GUSTAVSON,P. V., AOAR,HILDA,D. & CRaMER,DOROTHYI. (1954). Amer. J. trop. Meal. Hyg., 3, 1008. JAMES, S. P., NICOL, W. D. & SHUTE, P. G. (1933). Parasitology, 25, 87. LUDVIK, J. (1956). Zbl. Bakt., I. Abt., Orig., 166, 60. - (1958). Ibid., 172, 330. --(1962). Proceedings qf the First International Congress of Protozoology. Prague (in press). MICHAELIS,L. (1931). Biochem. Z., 234, 139. ROBEaTSON, J. D. (1960). In: Bargman, W., Peters, D. & Wolpers, C. (Ed.). Proc. 4th Int. Conference on Electron Microscopy, 1958, vol. 2, 159. Berlin, Gottingen & Heidelberg: SpringerVerlag. S H U T E , P. G. (1946). Trans. R. Soc. trop. Med. Hyg., 40, 189. ,

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