Cell Tissue Res (1986) 245:281 290
a n d T'tssue
Structure and development of the surface coat
of erythrocytic merozoites of Plasmodium knowlesi
L.H. Bannister 1, G.H. Mitchell 2, G.A. Butcher 2 *, E.D. Dennis 2, and S. Cohen 2
Departments of 1Anatomy and 2Chemical Pathology, Guy's Campus,
United Medical and Dental Schools of Guy's and St. Thomas's Hospitals, London, Great Britain
Summary. The surface of extracellular merozoites of P.
knowlesi is covered with a coat 15-20 nm thick, made up
of clusters of filaments standing erect on the plasma mem-
brane. Filaments have stems 2 nm thick, the peripheral ends
of which are complex, branching or ending in long trailing
threads. Coat filaments occur on the surface of the parasite
in regular rows at an early schizont stage, and persist until
well after merozoite release. They are sensitive to trypsin
and papain, and bind ethanolic phosphotungstate, indicat-
ing a proteinaceous nature. They are also removed by expo-
sure to phosphate-buffered saline. Filaments bear negative
charges, binding cationised ferritin throughout the depth
of the coat and staining with ruthenium red. They cover
the whole merozoite surface and mediate intercellular adhe-
sion at distances of 15 150 rim, membrane to membrane.
It is suggested that these filaments correspond to a major
merozoite surface protein, and are important in the initial
capture of red cells.
Key words: Electron m i c r o s c o p y - Malaria parasites - Mer-
ozoites Surface coat Maturation Plasmodium knowlesi
Electron-microscopic studies of various species of Plasmo-
dium have shown that merozoites are covered in thick fuzzy
coats by means of which they adhere to red cells prior
to their invasion, the coat being lost during the process
of red cell entry (Ladda et al. 1969; Bannister et al. 1975;
Aikawa etal. 1978; Aikawa and Miller 1983; Langreth
et al. 1978; Miller et al. 1979). Its removal in P. knowlesi
with trypsin or treatment with immune sera which bind
to coat components (Miller et al. 1975) inhibit invasion,
as do some monoclonal antibodies directed against the mer-
ozoite surface (Epstein et al. 1981 ; Deans et al. 1982, 1984;
Miller et al. 1984). Several major immunological studies
have now described various parasite antigens at the mero-
zoite surface, at least some of which are likely to be coat
components (Mason et al. 1977; Langreth and Reese 1979;
Epstein et al. 1981 ; Miller et al. 1984).
The merozoite surface has some properties atypical of
a surface glycocalyx, for example, exposed sialic acid and
Send q[J))rint requests to: Dr. L.H. Bannister, Department of Anat-
omy, Guy's Campus, United Medical and Dental Schools, London
SEI 9RT, Great Britain
* Present address." Dr. G.A. Butcher, Department of Zoology,
Australian National University, Canberra, ACT., Australia
Cell
9 Springer-Verlag 1986
other lectin-binding carbohydrates have been found scarce
or missing in merozoites of P. berghei (Seed et al. 1974;
Seed and Kreier 1976), P. chabaudi (David et al. 1978) and
P. knowlesi (Mitchell et al. 1975; Bannister etal. 1977).
Merozoites of P. falciparum also carry a smaller negative
charge than do red cells (Heidrich et al. 1982).
However, some important questions still remain to be
answered. It is still uncertain whether the coat of free mero-
zoites of Plasmodium is an organized structural feature of
the parasite, or if, instead, it represents antigenic material
derived, e.g. from the parasitophorous vacuole contents,
adsorbed on the exterior of the cell (see e.g. Mason et al.
1977; Aikawa and Seed 1980). This doubt is related to
the reportedly rather unstructured form of the coat in free
merozoites (Miller et al. 1975), and to its apparent absence
from the surfaces of merozoites within schizonts except
after the host cell has haemolysed immediately before re-
lease (Seed et al. 1976; Aikawa and Seed 1980). In exoeryth-
rocytic stages, free P. yoelii merozoites (Garnham et al.
1969) and developing merozoites of P. berghei (Desser et al.
1972) are known to have a complex surface substructure
as are free erythrocytic merozoites (Seed et al. 1974) al-
though the precise nature of these structures has not been
explored.
In the present paper we have investigated these prob-
lems by examining the detailed ultrastructure of the surface
both of free merozoites and of schizonts of P. knowlesi
at different stages of development, using a variety of fixa-
tives and cytochemical methods. Our results indicate that
the cell coat is indeed a regularly structured component
of the merozoite surface and that its formation commences
early in the erythrocytic schizont.
Materials and methods
Morphology o f parasites
P. knowlesi merozoites were harvested by sieving, from ma-
ture schizont-infected rhesus monkey blood (see Dennis
et al. 1975), and were collected in Medium 199 (Wellcome
Reagents). These were centrifuged into a pellet and immedi-
ately resuspended in glutaraldehyde (2.5% in either 0.1 M
cacodylate buffer (pH 7.3) containing 5 m M CaClz and
0.1 M sucrose, or in 0.1 M phosphate buffer (pH 7.3) with
0.1 M sucrose for 1-2 h at 4 ~ or at room temperature.
In one experiment mature schizonts were fixed in 2.5%
cacodylate-buffered glutaraldehyde containing 0.4 M su-
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 283

crose to cause osmotic shrinkage. After several washes in
the a p p r o p r i a t e buffer, cells pelletted by centrifugation were
treated with 1% buffered osmium tetroxide, then, in the
case o f cacodylate-buffered material, immersed for 1 2 h
at 4 ~ C in 0.5% aqueous uranyl acetate. After dehydration
in ascending concentrations o f acetone, pellets were embed-
ded in T A A B epoxy resin, and sections contrasted with
a saturated solution o f uranyl acetate in 50% ethanol
(10 min) and 0.4% lead citrate in 0.1 M N a O H (8 rain).
To d e m o n s t r a t e the surface coat more clearly, some sections
were stained in the uranyl acetate solution for 15 20 min,
then lead citrate for 10 rain.
To examine attachment to erythrocytes, freshly har-
vested merozoites were incubated for 45 s or 2 min with
fresh rhesus m o n k e y cells, after which fixation was as de-
scribed above (for details o f this procedure, see Bannister
et al. 1975). Developmental studies were carried out on in-
fected b l o o d taken from the ear-lobe o f rhesus monkeys
with parasitaemias o f 15-30%. D r o p s o f blood containing
stages o f parasites ranging from late trophozoites to late
schizonts were suspended in either the glutaraldehyde-caco-
dylate fixative described above, or in 2.5% (v/v) glutaralde-
hyde in 0.1 M p h o s p h a t e (pH 7.0) containing 1% or 4%
(w/v) tannic acid with 0.5 mg/ml saponin. After 3 h fixa-
tion, cells were washed in 0.1 M p h o s p h a t e buffer, treated
with 1% buffered osmium tetroxide for 2 h and centrifuged
pellets were d e h y d r a t e d and embedded as before. Smears
were also m a d e o f infected blood, in parallel, stained with
Giemsa, and viewed by light microscopy.
Cytochemical studies
Proteins. To study the sensitivity of the surface coat to
proteases, suspensions o f merozoites (10 9 m l - 1) were incu-
bated with 0.1% (w/v) trypsin, or 0.05% (w/v) papain in
phosphate-buffered saline (PBS) at p H 7.4, or in PBS alone
for various times (5-25 min). To stain proteins selectively,
pelletted merozoites were fixed for 1 h in 2.5% glutaralde-
hyde (phosphate-buffered), washed in buffer but not treated
with osmium, then d e h y d r a t e d in ascending concentrations
of ethanol with two changes o f 100% ethanol, and stained
with 1% ethanolic phosphotungstate for 1-2 h (Bloom and
A g h a g a n i a n 1966). Pelleted cells were washed in ethanol,
cleared in e p o x y - p r o p a n e and embedded in resin. Sections
were viewed without further staining.
Fixed negative surface charges
Ruthenium red staining (Luft 1971 ; Seed et al. 1974): cells
were fixed for 1 h at r o o m temperature in the dark in 2.5%
glutaraldehyde (cacodylate-buffered at p H 7.2), containing
0.5 mg m1-1 ruthenium red (Emscope Ltd). Resuspended
cells were then washed in cacodylate-buffer and treated for
2 h in the d a r k at 4 ~ C with cacodylate-buffered 1.0% os-
mium tetroxide, ruthenium red being a d d e d to 0.5 mg m l - 1
in both these solutions. Cationised ferritin binding (Danon
et al. 1972; King and Preston 1977): cells were fixed at
r o o m temperature for 15 rain 1 h in 2.5% glutaraldehyde
in phosphate buffer, washed three times in buffer then
treated with 1% glycine in phosphate buffer to neutralise
aldehyde-induced negative charges. Suspensions o f a b o u t
109 cells ml 1 were then exposed for 30 min to cationised
ferritin (Miles Ltd) at a concentration of 2.3 mg ml 1. The
cells were washed three times in phosphate buffer and fixed
for a further 30 rain in 1% buffered glutaraldehyde, treated
for 2 h with 1% buffered osmium tetroxide and prepared
for sectioning. Control merozoites were treated in the same
way, except that normal ferritin ( G r a d e VI, Sigma Inc) was
substituted at the same concentration for the cationised
type.
Results
Structure of the surface coat in free merozoites
Seen in section, the surface coat is formed largely o f tooth-
like projections a b o u t 18 20 nm tall, with expanded apices
(Figs. 1 7). In tangential sections o f the surface (Figs. 8,
9) the projections are seen as irregular polygonal masses
which together create a reticulum covering the whole mero-
zoite surface, including the apex. A conspicuous cluster o f
these structures arises from a small hillock protruding api-
cally over the c o m m o n duct o f the rhoptry canals (Figs. 2,
3). Over the cytostome the coat is formed by a continuous

Fig. I. Longitudinal section of a P. knowlesi merozoite showing the bristly appearance of the cell coat which covers the entire surface.
In this example, the parasite is attached apically to a red cell by a few long thread-like projections, x 49350

Fig. 2. Longitudinal section through the apex of a merozoite. The clumps of coat material are regularly spaced over the surface, including
the apical mound overlying the rhoptry canal (arrowhead). • 219000

Fig. 3. A merozoite apex showing prominent coat projections, including a conspicuous cluster on the apical mound (compare Fig. 2).
Some coat projections also form connections with another merozoite (left). • 100000

Fig. 4. Section through the merozoite surface (non-apical) after tannic-glutaraldehyde fixation, showing the three membranes of the
pellicle and dense clumps of coat material on the plasma membrane, x 350000

Fig. 5. Section through the merozoite surface, heavily stained to show thread-like extensions emerging from the tips of coat projections
(e.g. arrows'), x 320000

Figs. 6, 7. A section through the merozoite surface, heavily stained to show details of coat substructure and photographed at two
focal positions to clarify ultrastructural detail. Fig. 6 is a slightly underfocused image, and Fig. 7 is at or close to exact focus. The
underfocus image has relatively high contrast, though also containing some background interference; indicated are thin projecting
filaments, some of them apprently branched (arrows), and an example of a long trailing filament lying along the cell surface (arrowhead).
In Fig. 7, background interference is reduced and although the finer details are not clear because of the poorer contrast, the same
general structures are also visible. • 337000
284
 285

layer o f the same thickness. Tangential and vertical sections
through the cell surface indicate that each projection is real-
ly a group o f 5-10 parallel filaments each a b o u t 2 nm thick
and 18-20 nm tall, attached basally to the merozoite's plas-
m a m e m b r a n e (Figs. 5-8, 26). A t their apices each group
has a more complex but ill-defined structure; in thin, heav-
ily stained sections individual filaments a p p e a r to have ex-
p a n d e d or bifurcate tips (Figs. 6, 7). Some filaments are
much longer (Figs. 5-7), extending up to 150 nm; these ei-
ther project outwards from the coat (Fig. 5) or lie parallel
to the cell surface (Figs. 6, 7). Similar strands connect mero-
zoites with red cells during early stages o f attachment
(Fig. 13), and are also involved in merozoite-platelet or
merozoite-merozoite adhesion (Figs. 3, 16); they occur over
the whole merozoite surface including the apex, and are
often associated with substantial bending of the attached
red cell (Fig. 13).
Long filaments, p r o b a b l y o f this type, also connect mer-
ozoites with other cells (see below).
Cytochemistry
The coat projections stained with ethanolic p h o s p h o t u n g -
state (Fig. 10), and were completely removed by treatment
of cells with 0.1% trypsin or 0.05% p a p a i n for 5 rain at
37 ~ C. In controls incubated at the same temperature in
PBS the surface coat was still present at 10 min, but except
for a few short filaments, had disappeared by 20 min. Mer-
ozoites incubated for prolonged periods (40 min at 37 ~ C)
in M e d i u m 199 likewise lost their coats.
Cationised though not uncharged ferritin was b o u n d
by the whole merozoite surface. M o r e detailed inspection
showed that granules were sited uniformly at all levels with-
in the merozoite coat, including the outer surface o f the
plasma m e m b r a n e (Fig. 11). Ruthenium red (Fig. 12)
stained the outer surface o f the plasma m e m b r a n e strongly
to give a thin (3 nm) continuous dense coating, and was
also b o u n d to the projecting coat filaments, though less
prominently. Inclusion o f ruthenium red in the fixative al-
ways caused some damage to merozoites (but not to eryth-
rocytes), which either disintegrated or were penetrated by
the dye to give some electron density to the deeper layers
of the pellicle, as also shown in Fig. 12. Incubation in ruthe-
nium red before fixation resulted in similar damage.
Attachment to other cells
Merozoites in early stages o f adhesion to red cell (fixed
at 45 s) show long (up to 150 nm) threads connecting the
two surfaces (Fig. 13), as well as closer contacts (20 nm:
see Fig. 14). The more distant attachments are formed over
the whole surface including the apex, but a p p e a r to be much
less numerous than the closer, 20 nm type. Such long at-
tachments often a c c o m p a n y substantial bending o f the at-
tached red cell (Fig. 13). As found by A i k a w a et al. (1978),
even closer (about 8 n m ) j u n c t i o n a l (invasion initiating)
contacts, not shown in the present study, are made only
with the apical region of the parasite. Filamentous attach-
ments 20~! 50 nm long were also seen between merozoites
and other cells including neighbouring merozoites (Fig. 3)
and platelets (Figs. 15, 16) by contacts o f the medium
(20 nm) and long range (up to 150 nm) types, but no close
junctions have been seen in such cases. Such contacts oc-
curred in samples which had been pelletted at relatively
low speeds (see Materials and methods) so that cells were
not closely packed and did not show any signs of having
been forced into contact with each other.
Coat development
In the earlier stages o f the erythrocytic cycle (ring, tropho-
zoite, earliest schizont), the surface o f the parasite is closely
a p p o s e d to the p a r a s i t o p h o r o u s vacuole membrane, as de-
scribed by Wunderlich et al. (1982a, b), and there is no
detectable coat on the parasite's surface. The earliest signs

Fig. 8. Tangential section through the surface of a merozoite, showing the reticular appearance of the coat. x 142000

Fig. 9. Detail of Fig. 8. The coat is seen to be composed of clumps of small dots corresponding to cross sections or perpendicular
views of filaments, x 275650

Fig. 10. Perpendicular section through the surface of a merozoite stained only with ethanolic phosphotungstate. Visible on the surface
are some coat projections. The membranes of the deeper layers of the pellicle appear in negative contrast, x 84000

Fig. 11. A merozoite surface after incubation in cationised ferritin. The grains of ferritin are distributed at all levels in the cell coat.
x 187000

Fig. 12. The surface of a merozoite fixed in ruthenium red-glutaraldehyde. The dye has bound to the coat projections, but also forms
a prominent continuous layer immediately adjacent to the plasma membrane. As mentioned in the text, the dye has also penetrated
the cytoplasm to outline other structures within the cell. x 187000

Fig. 13. An early stage of merozoite attachment to a red cell (45 s incubation) showing long thin strands connecting the two cell
surfaces at distances of 25 120 nm. Note the abrupt curvature in the red cell surface (above) typical of many such interactions. In
this example a second red cell is also attached by a few strands to the base of the parasite (below). x 57360

Fig. 14. Medium range (20 nm, membrane to membrane) attachment between a merozoite and a red cell, involving large numbers
of coat filaments, x 96000

Fig. 15. Contact of the medium-range type between the apical prominence of a merozoite and a platelet. No signs of invasive activity
are visible, x 72000

Fig, 16. Attachment between a merozoite and a platelet. A long distance type of connection is indicated by the arrow, x 72000
286

Fig. 17. Tangential section through the surface of an early schizont within a red cell, fixed in tannic-glutaraldehyde. The newly formed
cell coat is visible as continuous lines of dense material. The section has also cut into an elongated nucleus (nu) close to the surface.
x 128850
Fig. 18. Section cut perpendicular to the surface of an early schizont showing dense tooth-like projections of the newly formed coat.
The parasitophorous vacuole membrane (white arrowhead) is spaced 25 nm from the schizont plasma membrane except where the
coat is absent (arrow). Endoplasmic reticulum, some with attached ribosomes, is also visible. Tannic-glutaraldehyde fixation, x 100000
Fig. 19. High power view of a tangential section of an early schizont surface showing two rows of coat material (arrows). Each row
is composed of dense granules (e.g. arrowheads'), corresponding to coat filaments (compare Figs. 20, 21). Tannic-glutaraldehyde fixation.
x 470 000
 287

Fig. 23. Section through a late schizont showing the apical half of a merozoite and part of another merozoite (right). The bristly
coat is present on the merozoite surfaces, and is clearly visible even where the flocculent material within the parasitophorous vacuole
(asterisk) overlies it. The membranes of adjacent merozoites (zone indicated by arrows) are separated by a double layer of coat material
(compare Fig. 25). Tannic acid-glutaraldehyde fixation, x 56200

Fig. 24. Section through the apex of a newly formed merozoite still within a red cell. The parasitophorous vacuole membrane (pvm)
is separated by about 25 nm from the merozoite plasma membrane (mpm), the the projections of the merozoite coat can be seen
covering the whole merozoite apex. Note also the inner pellicular membranes and polar rings (arrowheads) of the merozoite. Tannic
acid-glutaraldehyde fixation, x 117400

Fig. 25. Section through a developing cleft separating two merozoite buds. Two layers of coat projections, one on either of the lining
membranes are visible, separated by a narrow electron-lucent corridor. Tannic acid-glutaraldehyde fixation, x 314000

of coat formation occur at the two nucleus stage of schizont
development (Figs. 17, 18).
The coat first appears as patches of regular denticula-
tions on the parasite's surface, the parasitophorous vacuole
m e m b r a n e retreating to a distance of about 20 n m in such
areas (Figs. 18, 22, 26). As development proceeds, more
of these coated regions form, grow in size and eventually
coalesce to cover the whole surface, including the fissures
which by this time are delimiting the budding merozoites
(Figs. 23-25) and also the residual body as this is formed.
In tangential sections of the early coat, clumps of filaments
are arranged in regular, almost continuous rows (Fig. 17),
but immediately prior to release of mature merozoites, this
pattern changes to the less regular appearance typical of
free merozoites.
Detailed examination of the coat of early schizonts, us-
ing very thin (gray), heavily contrasted sections, shows the
coat to be composed of groups of filaments with dimensions
similar to those of free merozoites (Figs. 19-21) although
their apices are not obviously branched; however, their
apices appear thicker than their stems, suggesting the pres-
ence of parallel branches or other complexities (see Fig. 26).
Their bases also often appear thicker than the filament
stems (Figs. 20, 21). Tangled masses of threads seen in

Figs. 20, 21. Thin, heavily stained sections cut perpendicular to the parasite's surface, showing individual filaments (arrowheads). Note
that the filaments have complex apparently expanded or branched tips. Fig. 20 is from an early schizont, and Fig. 21 from an early
merozoite bud. Tannic-glutaraldehyde fixation. Both x 500000

Fig. 22. Section through the plasma membrane and newly formed coat of an early schizont and the overlying parasitophorous vacuole
membrane (pvm). The individual projections have a bushy appearance indicating the presence of numerous filaments within the section's
thickness. Longer filaments, some of them curved (arrowhead) emerge from a few of the projections. Dense material also adheres
to the inner aspect (its underside in this figure) of the parasitophorous vacuole membrane. Visible within the parasite's cytoplasm
are the two membranes of the nuclear envelope and portion of the nucleus (nu). Tannic acid-glutaraldehyde fixation. • 600000
288
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Fig. 26. Diagrammatic summary and interpretation of some of the
chief results of this paper. A, B The form of coat projections situ-
ated on the plasma membrane of the mature merozoite, seen in
section of routine thickness (A) and an ultrathin section (B) show-
ing the proposed filamentous composition of the projections with
branched and trailing terminals (compare with Figs. I 7, 21). C,
D Distribution of ruthenium red (C) and cationised ferritin bind-
ing. E, F, G The different types of merozoite-red cell contact, in-
cluding long-range (E), medium range (F) and close-range contacts,
in relation to the size of coat projections. H The relationship be-
tween the membranes of the parasite and the parasitophorous va-
cuole in a very early schizont where the membranes are apposed,
and after coat formation when the two are separated by the project-
ing clumps of coat filaments (compare with Figs. 18 and 22). I
The relationships between the surface of two merozoites within
a late schizont, when a double row of projections separates the
two plasma membranes (see Fig. 25)
thicker sections to emerge from some projections (Fig. 22)
perhaps correspond to the long extended type of mature
filament.
Where, during development, two merozoite surfaces are
juxtaposed (Figs. 23, 25), the plasma membranes of the
neighbouring cells are separated by at least 40 nm (i.e. the
combined thicknesses of the two coats: see Fig. 26), even
when infected cells have been shrunk by treatment with
hypertonic fixative (see Materials and methods) to force
the merozoites together. Likewise, the minimum distance
of 20 nm between parasite membrane and parasitophorous
vacuole membrane is maintained at all stages; it is note-
worthy that the parasitophorous vacuole membrane also
shows signs of some type of coating on its inner (vacuolar)
side, consisting of intermittent clumps of densely staining
material (Fig. 22).
It should be emphasised that the parasite's coat persists
through the late stages of merozoite detachment within the
parasitophorous vacuole, although after routine glutaralde-
hyde fixation it is often somewhat obscured by the floccu-
lent granular material which fills the vacuole at this stage
".....
.:~.,..~,
-
(see also Seed et al. 1976; Aikawa and Seed 1980). However,
it is clearly visible in tannic acid-glutaraldehyde fixed cells
at all stages of merozoite maturation (Figs. 23, 24), differing
in its early stages from the coat of freshly released mero-
zoites only in the details of its pattern of distribution (see
above).
Discussion
The results of this study indicate that the merozoite coat
is an integral part of the parasite's surface, with a highly
ordered structure first established in the early schizont, and
extending to cover the entire schizont plasma membrane
well before merozoites begin to be formed (see Fig. 26).
The detailed interpretation of the electron micrographs
depends, of course, on assumptions about preparative tech-
niques and observations made near the limits of useful bio-
logical microscopy. However, the structures described here
were seen with more than one method of fixation, maximum
clarity being provided in tannic acid-treated cells. Whether
this method generates important artefacts is not certain,
but its use in studies of other cells indicates that it is a
valuable means of visual~ filamentous protein assemb-
lies. The highly ordered state of the coat filament arrays,
and the constancy of their form both inside schizonts and
on free merozoites shows that they are not, for example,
artefacts generated by precipitation of soluble proteins on
to the cell surface. Microscopy of such small structures is
obviously fraught with problems, and the descriptions given
here, although the result of observations of many examples
sectioned in various planes and photographed at different
levels of focus and contrast, are only tentative.
Individual filaments are anchored to the membrane
strongly enough to maintain attachment to red cells and
to mediate considerable bending forces, although they al-
low re-orientation of the merozoite on the red cell, or even
detachment, if effective apical contact is not made (see also
Dvorak et al. 1975; Miller et al. 1979). The results suggest
that two types of filament exist, a shorter (about 20 nm)
type which forms medium-range contacts and a long (per-
haps up to 150 nm) class responsible for more distant adhe-
sions, although the shorter form may represent longer fila-
ments which have shed their ends. Closer contact (not stud-
ied here, see Fig. 26) which occurs only on the merozoite
apex and precedes invasion, does not seem to be associated
with either of these types of filament. On all parts of the
merozoite, including the apex, both types of filament are
able to interact, apparently non-specifically, with a range
of cellular surfaces, e.g. of platelets and other merozoites
as well as red cells.

Filaments are proteinaceous, according to their com-
plete removal by trypsin and papain, and p r o b a b l y contain
appreciable a m o u n t s o f basic amino acid residues, since
they bind ethanolic phosphotungstate (Bloom and A g h a -
ganian 1966). They a p p e a r to be covered in fixed negative
charges since they bind ruthenium red and cationised ferri-
tin. Such charges m a y be borne by polyanionic carbohy-
drates, as in Trypanosoma lewisi (e.g. Dwyer 1975) al-
though, in merozoites, glycosylation is not evident because
they lack exposed lectin binding sites.
The chemical identity o f the coat filaments can only
be surmised, but their sizes and numbers indicate that they
constitute one or more quantitatively m a j o r surface molecu-
lar species. F o r P. knowlesi, several merozoite surface com-
ponents have been described and partially chemically char-
acterised. A number of these derive from a c o m m o n precur-
sor molecule synthesised by ~ 4 nuclei schizonts as a glyco-
protein with a molecular weight of 230 kilodaltons (kDa)
(Epstein et al. 1981) which is processed to yield fragments
o f 75, 57, 50 and 43 k D a at merozoite release. Of these,
only the 43 k D a fragment is glycosylated (David et al.
1984). A n o t h e r antigenically distinct class, also first synthe-
sised in immature schizonts some hours before rupture, is
a 143 k D a protein which is processed at merozoite release
to a 140 k D a protein (Miller et al. 1984; David et al. 1985).
A further pair o f mature merozoite surface proteins, with
molecular weights o f 44 and 42 kDa, are fragments of a
68 k D a protein which first appears in late schizonts (seven
or m o r e nuclei), and is processed to the smaller molecules
at schizont rupture (Deans et al. 1984). These groups o f
surface c o m p o n e n t s have been recognized, and in some
cases isolated, using monoclonal antibodies. The distinct
technique o f surface radio-iodination has also been applied
fruitfully to P. knowlesi merozoites and confirms the pres-
ence o f at least eight components, distinguished by molecu-
lar weights in the range 22-140 k D a (Johnson et al. 1981).
Faced with this range o f possible candidates, it may
be noted that the filaments are sufficiently small to repre-
sent single or small groups o f protein molecules. As a rough
comparison, for example, molecules of the filamentous
muscle protein p a r a m y o s i n have a similar size (2 nm thick
and 140 nm long) and a molecular weight o f 220 k D a
( M c C u b b i n and K a y 1968).
Can we then propose an identity for the coat filaments ?
The timing o f the first appearance of the coat appears to
rule out the 68 k D a protein, and to favour either the
230 k D a or 143/140 k D a species. The lack o f a marked
change in filament structure between the schizont and free
merozoite stages found in the present study suggests that
the latter (the 143/140 k D a protein of Miller et al. 1984
and David et al. 1985) is the more likely candidate, since
this alters only slightly in molecular weight during develop-
ment, whereas the former is cleaved extensively at matura-
tion. The 143/140 kDa, but not the 230 k D a molecule, is
also removed by gently trypsinisation, as judged by radio-
iodination (Johnson et al. 1981), like the observed coat fila-
ments.
It is also noteworthy that there are ultrastructural signs
of other molecular species present at the cell surface, e.g.
the dense ruthenium red-positive coating of the outer leaflet
o f the plasma membranes shown in the present study.
Clearly much remains to be done to satisfactorily correlate
the structure and biochemistry o f the merozoite surface in
P. knowlesi and in other species o f Plasmodium.
289
Acknowledgements. The authors wish to thank the Medical Re-
search Council and the World Health Organization for their finan-
cial assistance during this study.
References
Aikawa M, Miller LH (1983) Structural alteration of the erythro-
cyte membrane during malarial parasite invasion and intra-
erythrocytic development. In : Evered D, Whelan J (eds) Malar-
ia and the red cell (Ciba Symposium 94), pp 45 63. Pitman,
London, pp 45 63
Aikawa M, Seed TS (1980) Morphology of Plasmodia. In: Kreier
JP (ed) Malaria. Vol 1. Academic Press, New York, pp 285 344
Aikawa M, Miller LH, Johnson J, Rabbege J (1978) Erythrocyte
entry by malarial parasites. A moving j unction between erythro-
cyte and parasite. J Cell Biol 77 : 72 82
Bannister LH, Butcher GA, Dennis ED, Mitchell GH (1975) Struc-
ture and invasive behaviour of Plasmodium knowlesi merozoites
in vitro. Parasitology 71:483-491
Bannister LH, Butcher GA, Mitchell GH (1977) Recent advances
in understanding the invasion of erythrocytes by merozoites
of Plasmodium knowlesi. Bull WHO 55:163 169
Bloom FE, Aghaganian GK (1966) Cytochemistry of synapsis:
selective staining for electron microscopy. Science
154:1575-1577
Danon D, Goldstein L, Marikowsky Y, Skutelsky E (1972) Use
of cationised ferritin as a label of negative charges on cell sur-
faces. J Ultrastruct Res 38:50(~510
David PH, Hommell M, Benichou JC, Eisen HA, Pereira da Silva
LH (1978) Isolation of malaria merozoites: release of Plasmo-
dium chabaudi merozoites from schizonts bound to immobilized
Concanavalin A. Proc Natl Acad Sci USA 75:5081 5084
David PH, Hadley TJ, Aikawa M, Miller LH (1984) Processing
of a major parasite surface glycoprotein during the ultimate
stages of differentiation in Plasmodium knowlesi. Mol Biochem
Parasitol 11 : 267-282
David PH, Hudson DE, Hadley TJ, Klotz FW, Miller LH (1985)
Immunization of monkeys with a 140 kD merozoite surface
protein of Plasmodium knowlesi malaria: appearance of alter-
nate forms of this protein. J Immunol 134 : 41464152
Deans JA, Alderson T, Thomas AW, Mitchell GH, Lennox ES,
Cohen S (1982) Rat monoconal antibodies which inhibit the
in vitro multiplication of Plasmodium knowlesi. Clin Exp Im-
munol 49 : 297-309
Deans JA, Thomas AW, Alderson T, Cohen S (1984) Biosynthesis
of a putative protective Plasmodium knowlesi antigen. Mol Bio-
chem Parasitol 11 : 189 204
Dennis ED, Mitchell GH, Butcher GA, Cohen S (1975) In vitro
isolation of Plasmodium knowlesi merozoites using polycarbon-
ate sieves. Parasitology 71:475-481
Desser SS, Weller I, Yoeli M (1972) An ultrastructural study of
the pre-erythrocytic development of Plasmodium berghei in the
tree rat Thamnomys surdaster. Can J Zool 50:821-825
Dvorak JA, Miller LH, Whitehouse WC, Shiroishi T (1975) Inva-
sion of erythrocytes by malaria merozoites. Science
187:748 750
Dwyer DM (1975) Cell surface saccharides of Trypanosoma lewisi.
1. Polycation-induced cell agglutination and fine structure cyto-
chemistry. J Cell Sci 19:621 644
Epstein N, Miller LH, Kauschel DC, Udeinya 1J, Rener J, Russell
JH, Asofsky R, Aikawa M, Hess RL (1981) Monoclonal anti-
bodies against specific surface determinant on malarial (Plas-
modium knowlesi) merozoites block erythrocyte invasion. J Im-
munol 127:212-217
Garnham PCC, Bird RG, Baker JR, Killick-Kendrick R (1969)
Electron microscopic studies on the motile stages of malaria
parasites VIII. The fine structure of merozoites of exoerythro-
cytic schizonts of Plasmodium berghei yoelii. Trans R Soc Trop
Med Hyg 63:328 332
Heidrich H-G, Mrema JEK, Vander Jagt DL, Reyes P, Breckmann
KH (1982) Isolation of intracellular parasites (Plasmodium fal-
290
ciparum) from culture using free-flow electrophoresis: separa-
tion of the free parasites according to stages. J Parasitol
68:443-450
Johnson JG, Epstein N, Shiroishi T, Miller LH (1981) Identifica-
tion of surface proteins on viable Plasmodium knowlesi mero-
zoites. J Protozool 28:160-164
King CA, Preston TM (1977) Studies of anionic sites on the cell
surface of the amoeba Naegleria gruberi using cationised ferri-
tin. J Cell Sci 28:133-149
Ladda RL, Aikawa M, Sprinz H (1969) Penetration of erythrocytes
by merozoites of mammalian and avian malarial parasites. J
Parasitol 65:633-644
Langreth SG, Reese RT (1979) Antigenicity of the infected-erythro-
cyte and merozoite surfaces in Falciparum malaria. J Exp Med
150:1241-1254
Langreth SG, Jensen JB, Reese RT, Trager W (1978) Fine structure
of human malaria in vitro. J Protozool 25:443 452
Luft JH (1971) Ruthenium red and violet. I. Chemistry, purifica-
tion, methods of use for electron microscopy and mechanism
of action. Anat Rec 171 : 347-368
Mason SJ, Aikawa M, Shiroishi T, Miller LH (1977) Further evi-
dence for the parasitic origin of the surface coat on malarial
merozoites. Am J Trop Med Hyg 26:195 197
McCubbin WD, Kay CM (1968) The subunit structure of fibrous
muscle proteins as determined by osmometry. Biochem Biophys
Acta 154 : 239-241
Miller LH, Aikawa M, Dvorak JA (1975) Malaria (Plasmodium
knowlesi) merozoites: immunity and the surface coat. J Im-
munol 114 : 1237-1242
Miller LH, Aikawa M, Johnson JG, Shiroishi T (1979) Interaction
between cytochalasin-B-treated malarial parasites and erythro-
cytes. Attachment and junction formation. J Exp Med
149:172 184
Miller LH, David PH, Hudson DE, Hadley TJ, Richards RE,
Aikawa M (1984) Monoclonal antibodies to a 140,000-M.W.
protein on Plasmodium knowlesi merozoites inhibit their inva-
sion of rhesus erythrocytes. J Immunol 132:438442
Mitchell GH, Butcher GA, Cohen S (1975) Merozoite vaccination
against Plasmodium knowlesi malaria. Immunology 29 : 397-407
Seed TM, Kreier JP (1976) Surface properties of extracellular ma-
laria parasites: electrophoretic and lectin-binding characteris-
tics. Infect Immun 14:1339-1347
Seed TM, Aikawa M, Sterling CR, Rabbege JR (1974) Surface
properties of extracellular malaria parasites: morphological and
cytochemical study. Infect Immun 9:750-761
Seed TM, Sterling CR, Aikawa M, Aikawa M, Rabbege J (1976)
Plasmodium simium: Ultrastructure of erythrocytic phase. Exp
Parasitol 39 : 26~276
Wunderlich F, Stfibig H, K6nigk E (1982a) lntraerythrocytic de-
velopment of Plasmodium knowlesi: structure, temperature- and
Ca + +-response of the host and parasite membranes. J Proto-
zool 29 : 49-59
Wunderlich F, Stfibig H, K6nigk E (1982b) Development of Plas-
modium chabaudi in mouse red blood cells: structural properties
of the host and parasite membranes. J Protozool 29 : 60-66
Accepted February 16, 1986