The surface of the malaria parasite

EXPERIMENTAL

PARASITOLOGY

36, 131-142

The Surface I. Scanning

( 1974)

of the Malaria

Electron

Microscopy

Parasite

of the Oocyst 1

C. P. A. STROME AND R. L. BEAUWIN Experimental Parasitology Division, Naval Medical Research Institute, National Naval Medical Center, Bethesda, Maryland 20014 (Submitted

for publication

August

2, 1973)

STROME, C. P. A., AND BEAUDOIN, R. L. 1974. The surface of the malaria parasite. 1. Scanning electron microscopy of the oocyst. Experimental Parasitology 36, 131-142. Scanning electron microscopy has been used to study surface characteristics of the early sporogonic stages of Plasmodium gaUinaceum and Plasrrwdium berghei. Observations upon oocysts from g-day old infections of P. gallinaceum in A&es aegypti and 14-day old infections of P. berghei in Anopheles stephewi indicate the oocyst surface is relatively smooth, although an outline of the underlying sporozoites can easily be recognized in the mature oocyst. Although all infections with each species were the same age, the stage of development of each oocyst appeared highly variable even within an individual mosquito. Oocysts appear to be covered by the overlying basement membrane which separates them from direct contact with the hemolymph as well as hemocytes. #Small buds designated as satellite ‘bodies were often seen attached to large oocysts of P. gallinuceum. Neither their origin nor their significance is yet known. During the study, numerous observations were made of the liberated sporozoites of both species. In each species the sporozoites are comma-shaped; however, those of P. gallinuceum are shorter, more strongly curved and stouter than those of P. berghei. INDEX DESCRIPTORS: Plasmodium gallinuceum; Plasm&urn berghei; Microscopy, Scanning Electron; Oocyst; Surfaces; Malaria; Critical Point Drying; Sporozoites; Anopheles stephensi; Aedes aegypti.

INTRODUCTION

croscopy to malaria parasites has provided a new dimension to our understanding of their biology. Use of this tool has successfully clarified many functional aspects which were previously poorly understood at best, and has extensively increased our knowledge of many of the vital processes of these parasites. Thus, a wealth of information is presently available on such diverse functions as the feeding process (Rudzinska and Trager 1957; Aikawa et al. 1966; Beaudoin and Strome 1972), differ-

The application of principles and techniques of modern transmission electron mi1 Supported by the Bureau of Medicine and Surgery Work Unit No. MR041.09.01.0127B6GJ and MF51.524.009.0032BB61. The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Navy Department or the naval service at large. The ducted Animal followed lication

experiments reported herein were conaccording to the principles outlined in the Welfare Act (PL 89-544 as amended) and the guidelines prescribed in DHEW PubNo. (NIH) 72-23, formerly PHS Publica-

tion No. 1024, “Guide Facilities and Care.” 131

Copyright All rights

0 of

1974

by Academic

reproduction in any

Press, form

Inc. reserved.

for

Laboratory

Animal

132

STROME

AND

BEAUDOIN

FIG. 1. Midgut of an uninfected female Aedes aegypti still slightly distended from a blood meal. The esophagus lies to the right. Both dorsal and ventral diverticula were removed during dissection. Just anterior to the point of its entry into the midgut proper is the exposed opening into the gut of one of the diverticula which was sheared off (arrow). Bands of circular muscles can be distinguished on the midgut surface, which is covered with a web of tracheae. Posteriorly, Malpighian tubules are attached to the ampulla ( X 135). FIG. 2. Midgut of an uninfected, unfed female Aedes aegypti. In the relaxed state, midgut muscle cells protrude noticeably (arrow). The specimen was photographed on a filter paper, the fibers of which can be seen in the background and will furnish in comparison some concept of the magnification ( X 162). entiation (Hepler et al. 1966; Aikawa 1966; Vanderberg et al. 1967; Beaudoin and Strome 1973), nuclear division ( Aikawa

and Beaudoin 1968; Aikawa et al. 1972) and sporogony (Terzakis et al. 1966; Gamham et al. 1969).

SCANNING

ELECTRON

MICROSCOPY

OF

THE

MALARIA

OOCYST

133

FIG. 3. Oocysts of Plesmodium gallinaceum on the haemocoel surface of the midgut of a female Aedes uegypti. Most of the oocyts visible are smooth spheres and are well preserved. Wrinkles on some specimens are artifacts of fixation, preparation or high energy scanning. In this micrograph, g-day old oocysts can readily be distinguished from the surface of the cells of the midgut. Extensive tracheation of the oocyst infected areas of the midgut can be seen. Smaller spheres are probably haemocytes which often adhere to the surface of oocysts ( X600).

On the other hand, transmission electron microscopy has been less productive in studying surface architecture and func-

tions which are surface dependent, as are *many important biological phenomena. In fact, very few attempts have been made to

134

STROME

AND BEAUDOIN

Oocysts in this micrograph show a tendency FIG. 4. Oocysts of Plasmodium gallinaceum. to stick to each other. This tendency to cluster may reflect the presence of some cementing substance or result simply from stretching of the covering basement membrane by enlarging oocysts on its inner surface ( X750).

study the surface structure of the malaria parasite. These attempts have dealt exclusively with the vertebrate phase of the

parasite life cycle using several different approaches, including negative staining ( Aikawa 1967), scanning electron micros-

SCANNING

ELECTRON

MICROSCOPY

OF THE MALARIA

OOCYST

135

FIG. 5. Mature oocyst of Plasmodimn g&inaceum. Folds can be seen in the membrane covering the two lower oocysts (arrows). Two oocysts on the left have collapsed, having probably ruptured during preparation for scanning. Notice the strands between the upper center oocyst and the collapsed ones below it. These strands appear to be derived from the covering membrane. The apparent smoothness of the outside surface of the oocyst capsule is striking for this magnification ( X 1700).

copy (Arnold et al. 1969; Schneider 1970)) and freeze etching (Meszoely et al. 1970, 1972; Seed et aE. 1971). The present study of early sporogonic development of the 8A Strain of Plasmadium gallinaceum in Aedes aegypti and the ANKA strain of Plasmodium berghei berghei in Anopheles Stephen& uses scanning electron microscopy and new, critical-

point drying techniques (Nemanic 1972; Cohen et al. 1968) to avoid distortion and overcome difficuhies encountered by earlier workers ( Sprinz 1969). MATERIALS AND METHODS

Nine-day old infections of the 8A strain of Plasmodium gallinaceum in Aedes aegypti and 14-day old infections of the

136

STROME

AND BEAUDOIN

FIG. 6. Oocyst of Phz.smodium gallinaceum. Small satellite cysts can frequently be seen attached to an oocyst, presumably attached by the basement membrane which lines the insect haemoco4 and covers the oocyst ( X 3600).

ANKA strain of P. berghei in Anopheles stephensi were used in the study. Infections were maintained as previously described (Beaudoin et al. 1974). Infected mosquitoes were dissected in .05 M phosphate buffer containing 1.25% glutaraldehyde v/v and 4% sucrose w/v at pH 7.2. Midguts were fixed in glutaraldehyde for 1 hr following dissection and dehydrated in an ethanol series from 20% through absolute alcohol in 10% increments at 30 min intervals, then into SO/SO v/v absolute alcohol/amyl acetate, into 100% amyl acetate and finally, criticalpoint dried in liquid CO,. Dried specimens were coated with 300 a of palladium gold and scanned with a JEOL JU3 scanning electron microscope.

RESULTS

Figure 1 is an electron micrograph of a normal, uninfected midgut of dedes cregypti. This mosquito had been recently fed, and the midgut remains slightly distended. The dorsal and ventral diverticula have been removed ~during dissection, exposing the opening of a diverticulum into the esophagus (arrow). At the posterior end of the midgut, Malpighian tubules can be seen just anterior to the pyloric ampulla. The surface of the distended midgut appears relatively smooth at this low magnification. However, the midgut of starved animals presents a different appearance, as shown in Fig. 2, with large bands of muscle cells seen protruding from the haemocoel surface. These protrusions may superficially

SCANNING

ELECTRON

MICROSCOPY

OF

FIG. 7. Mature oocysts of Phsmodium gallinaceum. of the oocysts. Their outline may be visible because inside of the capsule wall ( X 1700).

resemble immature oocysts in unfed specimens (Fig. 3). The micrograph in Fig. 3 illustrates the topography of the infected midgut. Oocysts at various stages of maturity cover the haemocoel surface. The tracheal system is particularly prominent in this picture and branches can be seen inter-

THE

MALARIA

OOCYST

137

Sporozoites are clearly visible in two of slight outward pressure against the

twining among the oocysts. The circular bands of cells seen bulging from the surface are muscle cells. Figure 4 is a higher magnification picture and shows oocysts apparently connetted to one another. The oocysts are covered by the membrane lining the

138

STROME

AND BEAUDOIN

of sporozoites is unmistakable and FIG. 8. Oocyst of Plusmodium gallinuceum. Outline presumably caused by the pressure they exert against the inside of the oocyst capsule, although even at this magn&cation the outer surface of the capsule appears relatively smooth (X.5250).

haemocoel which becomes tightly stretched over their surface and is responsible for this apparent bond between the individual oocysts. The oocysts in Fig. 5 illustrate the smoothness of the surface of the well-preserved specimen. This can be compared with the two collapsed cysts below it which did not survive the rigors of preparation. The covering membrane is revealed in this

micrograph as a fold (arrows) seen on the surface of two oocysts ‘on the bottom. Occasionally, large, full-sized oocysts can be seen attached to much smaller satellite cysts by the membrane which covers the outside of the oocyst capsule (Fig. 6). Sporozoites can be distinguished within the mature oocysts, as seen in Fig. 7. This effect probably results from very slight pressure caused by the mature sporozoites

SCANNING

ELECI’RON

MICROSCOPY

on the inner wall of the oocyst capsule. Figure 8 shows this same effect on another oocyst at a higher magnification. Free sporozoites can be observed lying on the surface of a P. gallinaceum oocyst in Fig. 9 and, in Fig. 10, several P. berghei sporozoites can be observed. The latter may have escaped from the ,ruptured capsules of oocysts in the center and bottom of the micrograph ( arrows ) . Large spherical cells which cluster around the oocysts in Figs. 9 and 10 appear to be hemocytes. DISCUSSION

Normally, the ookinete or zygote of the malaria parasite crosses the wall of the mosquito midgut to the haemocoel surface, where it transforms into an ‘oocyst. Once established on the haemocoel surface, the oocyst undergoes rapid growth followed by a highly organized orderly differentiation into a large number of sporozoites. The precise location of oocyst development and its relationship to the internal topography of the host midgut are not yet completely understood. Even the basic question of whether the oocyst is intracellular or extracellular has yet to be unequivocally resolved (Bafort 1971). Shute (1948) experimentally demonstrated that the location on the midgut where oocyst development occurs may vary along the anterior-posterior axis depending on the position assumed by the mosquito following its infective blood meal. Presumably, gravitational forces are in part responsible for these observations. Weathersby (1954; MO) showed, in a series of imaginative experiments, that the site of oocyst development is not vital to normal development of infective sporozoites. He injected young, sporogonic forms *directly into the haemocoel and these developed into sporozoites which produced infections when inoculated by the mosquitoes. Beaudoin et al. (1974) showed that, in natural infections of P. berghei in Anopheles stephensi, oocysts .develop in all layers of the gut

OF

THE

MALARIA

OOCYST

139

wall and that mature infective sporozoites commonly are released not only into the haemocoel but also into the lumen of the midgut. The present study is restricted to stages of oocyst development seen at 14 days after the mosquito host was infected with either P. gallinaceum or P. berghei. Although infected mosquitoes were maintained under uniform conditions of food, temperature, and humidity, the develop mental stage reached in this period of time by individual oocysts varied considerably in 8 single mosquito. Relatively immature forms were found side by side with mature forms i&d with fully formed sporozoites. Bafort (1971) calls attention to the wide range of oocyst sizes in P. oindcei at 14 days. Data provided by scanning electron microscopy suggest that, in both plasmodia studied, the oocysts which mature on the haemocoel surface of the midgut are covered and may be held in place by the basement membrane lining that cavity. In Figs. P7, several aspects of the overlying membrane are in evidence. This point has never been satisfactorily resolved using conventional light microscopy or transmission electron microscopy. Although our observations do not prove that every oocyst is covered in this way, we can nevertheless state that it is a common occurrence in oocysts of these two species. The surface of both the oocyst and sporozoite appears to be remarkably smooth in well-fixed specimens at the magnification obtained. The labyrinthine structure reported from transmission electron micrographs of both erythrocytic (Aikawa 1967) and exoerythrocytic merozoites (Beaudoin and Strome 1973) was not observed in sporozoites and if present in this stage is undoubtedly beyond the resolution of the instruments. Likewise, other surface structures reported for merozoites, including the polar apparatus ( = conoid) and cytostome, were not observed, presumably for the

140

STROME

AND

BEAUDOIN

gallinaceum. Comma-shaped sporozoite, free in the haemoFIG. 9. Oocyst of Plusmodium coel, is seen lying on the surface of an oocyst (x1700). FIG. 10. Plusmodium berghei. Several sporozoites are seen lying on the surface of oocysts. Sporozoites may have been released through the tears (arrows) visible in the capsules of the

SCANNING ELECXRON MICROSCOPY OF THE MALARIA

same reasons, although the cytostome appears to have been observed only rarely, at least in sporozoites of P. berghei (Garnham 1972), even with transmission electron microscopy. A comparison of sporozoites of P. gallinaceum with those of P. berghei in scanning electron micrographs reveals that, although both are comma-shaped, the gallinaceum sporozoite is shorter, stouter and more strongly curved. The surfaces of both forms appear very similar at this magnification. However, higher magnifications would be desirable to pursue studies of the surface of these very small stages. The satellite bodies which were observed as apparent buds from large gallinaceum oocysts have not been reported previously to our knowledge, and their biological significance is not known. ACKNOWLEDGMENTS The authors thank Mr. Fred Mitchell and Mr. T. A. Tubergen for their assistance in maintaining the life cycles of the parasites,

REFERENCES AIKAWA, M. 1966. The fine structure of the erythrocytic stages of three avian malarial parasites, Plasmodium fallax, P. Zophurae, and P. cathemerium. American Journu.! of Tropical Medicine and Hygiene 15, 449-471. AIKAWA, M. 1967. Ultrastructure of the pellicular complex of Plasmodium fallax. Journal of Cell Biology 35, 103-113. AIKAWA, M., AND BEAUDOIN, R. L. 1968. Studies on nuclear division of a malarial parasite under pyrimethamine treatment. Journal of Cell Biology 39, 749-754. AIKAWA, M., HEPLER, A. K., HUFF, C. G., AND SPRINZ, H. 1966. The feeding mechanism of avian malarial parasites. JournuE of Cell Biology 28, 355-373. AIKAWA, M., STERLING, C. R., AND RABBEGE, J, 1972. Cytochemistry of the nucleus of malarial parasites. In Basic Research in Malaria. Proceedings of the Helminthological Society of Washington (E. H. Sadun and A. P. Moon, Eds.), 39, 174-194, special issue.

OOCYST

141

ARNOLD, J. D., BALCERZAK, S. P., AND MARTIN, D. C. 1969. Studies on the red cell-parasite relationship. In Experimental Malaria. MiL itary Medicine (E. H. Sadun and A. P. Moon, Eds.), 134, 962-971, special issue. BAFORT, J. 1971. The biology of rodent malaria with particular reference to Plasmodium vinckei uinckei Rodhain, 1952. Annales de la So&t& belge MLdecine tropic& 51, l-204. BEAUDOIN, R. L., AND STROME, C. P. A. 1972. The feeding process in the exoerythrocytic stages of Plnsmadium lophurae based upon observations with the electron microscope. In Basic Research in Malaria. Proceedings of the Helminthological Society of Washington (E. H. Sadun and A. P. Moon, Eds.), 39, 163-173, special issue. BEAUDOIN, R. L., AND STROME, C. P. A. 1973. Plasmodium Eophurae: The ultrastructure of the exoerythrocytic stages. Experimental Parasitology 34, 313-336. BEAUDOIN, R. L., STROME, C. P. A., AND TUBERGEN, T. A. 1974. Plasmodium berghei berghei: The ectopic development of the ANKA strain in Anopheles stephensi. Experimental Parasitology ( in press ) . COHEN, A. L., MARLOW, D. P., AND GARNER, G. E. 1968. A rapid critical point method using fluorocarbons ( freons) as intermediate and transitional fluids. Journal de Microscopic 7, 331341. GARNHAM, P. C. C. 1972. Comments on plasmocha1 ultramicroscpy. In Basic Research in Malaria. Proceedings of the Helminthological Society of Washington (E. H. Sadun and A. P. Moon, Eds.), 39, 194-197, Special issue. GARNHAM, P. C. C., BIRD, R. G., BAKER, J. R., DESSER, ‘S. S., AND EL NAHAL, H. M. S. 1969. Electron microscope studies on motile stages of malaria parasites. VI. The ookinete of Plasnwdium berghei yoelii and its transformation into the early oocyst. Transactions of the Royal Society of Tropical Medic& and Hygiene 63, 187-194. HEPLER, P. K., HUFF, C. G., AND SPRINZ, H. 1966. The fine structure of the exoerythrocytic stages of Plasmodium fallax. The Journal of Cell Biology 30, 333-358. MESZOELY, C. A. M., BAHR, G., AND STEERE, R. 1970. Ultrastructural studies of Plasmodium gallinaceum. Journal of Parasitology 56, Sect. 2, Part 1, 236.

underlying oocysts. Sporozoites of this species appear curved than sporozoites of P. gallinuceum ( X 1600).

longer,

more slender

and less strongly

142

STBOME AND BEAUDOIN

MESLOELY, C. A. M., STEERE, R. L., AND BAHR,

G. F. 1972. Morphologic studies on the freeze-etched avian malarial parasite PZusmo&urn gallinaceum. In Basic Research in Malaria. Proceedings of the Helminthological Society of Washington (E. H. Sadun and A. P. Moon, Eds.), 39, 149-162, special issue. NEMANIC, M. K. 1972. Critical point drying cryofracture and serial sectioning. Scanning EZectron Microscopy 5, 297-304. RUDZINSKA, M. A., AND TRAGER, W. 1957. Intracellular phagotrophy by malaria parasites: An electron microscope study of Plasmodium lophurae. Journal of Protowology 4, 190-199. SCHNEIDER, M. D. 1970. SEM studies of malaria (Pkzsnwdium berghei yoelii) in mice. Scanning Electron Microscopy 3, 257-264. SEED, T., PFISTER, R., KREIER, J., AND JOHNSON, A. 1971. P&nodium gallinaceum: Fine structure by freeze etch technique. Experimental Parasitology 30, 73-81. SHUTE, P. G. 1948. The comparative distribution of oocysts of human malaria parasites on the stomach wall of Anopheles. Transactions of

the Royal Society of Tropical Medicine and Hygiene 42, 324. SPRINZ, H. 1969. Comments on “Studies on the red cell parasite relationship.” In Experimental malaria. MiZitary Medicine (E. H. Sadun and A. P. Moon, Eds.), 134, 976, special issue. TERZAKIS, J. A., SPFZNZ, H., AND WARD, R. A. 1966. Sporoblast and sporozoite formation in Plusnwdium gallinaceum infection of Aedes aegypti. In Research in Malaria. Military Medicine (E. H. Sadun and H. S. Osborne, Eds.), 131, 984-992, s’pecial issue. VANDERBERG, J., RHODIN, J., AND YOELI, M. 1967. Electron microscopic and histochemical studies of sporozoite formation in Plasmodium berghei. Journul of Protozoology 14, 82-103. WEATHERSBY, A. B. 1954. The ectopic development of malaria oocysts. Experimental Parasitology 3, 538-543. WEATHERSBY, A. B. 1960. Further studies on exogenous development of malaria in the haemocoels of mosquitoes. Experimental Parasitology 10, 211-213.