J. Cell Sd. I, 375-380 (1966)
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OBSERVATIONS ON SCALE PRODUCTION
IN PRYMNESIUM PARVUM
IRENE MANTON
Botany Department, University of Leeds
SUMMARY
Information is provided regarding certain structural and developmental aspects of the single
Golgi body present in the brown flagellate, Prymnesium parvum. Probably the most important
new observation is the demonstration of scale production within certain Golgi cisternae.
Different developmental states from immature to mature can be found in successive cisternae
on one side of the Golgi centre, the scales themselves being all similarly oriented with respect
to the inner and outer faces of the cisternae in which they are formed. A deep pit found in a
relatively constant position attached to the plasmalemma beside eachflagellarbase is interpreted
as being possibly the site of scale liberation. These findings are discussed in a preliminary way.
INTRODUCTION
The small brownflagellate,Prymnesium parvum Carter, is well known to biochemists
as the secretor of a potentially lethal fish poison. Thefinestructure has been examined
more than once by means both of the older methods involving osmic fixation with
methacrylate embedding (Manton & Leedale, 1963) and the newer methods combining glutaraldehydefixationwith Epon embedding (Manton, 1964a, c, d). The observations to be recorded here were obtained concurrently with those previously reported
(Manton, 1964c.fi) on the mitotic cycle and haptonema structure, respectively;
anyone wholly unfamiliar with this type of cell could usefully consult the first of these
two papers.
METHODS
Since the technical details have already been published it is sufficient to say here
that fixation was with an early formula for glutaraldehyde, namely as a 4% solution
neutralized with barium carbonate and made up with phosphate buffer without
sucrose. The relatively good quality of fixation achieved, in spite of the absence of
any special attention to the osmotic properties of the fixative, is in part explained by
the fact that the organism, which in life inhabits brackish waters, was maintained in
culture in a medium corresponding to half-strength sea water. Thefixativewas used
for 1 h, followed by i^-h washing in 3 changes of buffer. Post-osmication in 2 %
osmium tetroxide, also in phosphate buffer made up to pH 7, was applied for 2 h,
after which the material was embedded in Epon by standard procedures. Sections were
stained with lead citrate (Reynolds, 1963).
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OBSERVATIONS
The external morphology of the organism is sufficiently indicated by Fig. 1 which
illustrates two dried cells, each with a pair offlagellaand a short haptonema. The body
surface in life is covered with many small plate-scales with upturned rims, illustrated
previously from a whole mount in Manton & Leedale (1963) and visible here in various
planes of section in the ground beyond the cell surface in Figs. 2 and 3. Since the
structure of the flagellar bases and haptonema have already been made the subjects
of special study (Manton, 1964 a, d) it will be sufficient here to draw attention to the
various transverse and longitudinal sections of them included incidentally in Figs. 2,
3 and 5 and which are important as a means of clearly defining the region of the cell
with which the present observations are concerned.
As is well known (see especially Manton & Leedale, 1963) a good deal of the cell
volume in life is taken up by large membrane-bounded cavities with aqueous or
alcohol-soluble contents. After embedding, these cavities appear empty and most of
the more important organelles are accommodated within a compact area close to the
flagellar bases. Longitudinal sections through this region are reproduced in Figs. 2
and 3, with an oblique transverse section in Fig. 4.
Apart from the nucleus and laterally placed chloroplasts, which do not at present
concern us, the single Golgi body is the most conspicuous component. Elements of
rough endoplasmic reticulum bound the Golgi area on one side (see especially Fig. 3).
It is easy to prove continuity between such elements and the adjacent nuclear envelope, though not in the micrographs reproduced here. Continuity can, however,
reasonably be inferred in Fig. 3 (bottom, left) and is in any case sufficiently well known
not to need explicit demonstration here. Additional elements of endoplasmic reticulum are also present elsewhere in the cytoplasm and apposed to the chloroplast surface
in the manner explained elsewhere (Manton, 1964 c). The other organelles, including a
large mitochondrion partly surrounding the flagellar bases and with internal tubular
cristae strongly oriented towards them at the point of closest approach (see Fig. 2),
are also as previously described (Manton, 1964^).
Between the endoplasmic reticulum and the adjacent cisternae of the subtended
Golgi body (Fig. 3, left), the usual array of small vesicles is present, though in this
material there is as yet no information regarding their nature, direction of movement
or subsequent fate. These do not include any demonstrably separate vesicles covered
with the ' hairy' or ' alveolar' coating characteristic of many diverticula of various sizes
found attached to the edges and sometimes also to the flat faces of the Golgi cisternae
themselves. These diverticula appear to originate in this position and many of them
are retained throughout the developmental life of the cisterna, becoming if anything
slightly larger with age, though some may perhaps eventually become detached. There
is no evidence in this material of any developmental connexion between such coated
vesicles or diverticula and the endoplasmic reticulum.
Within the Golgi body, the cisternae are strongly asymmetrical and also heterogeneous. Both properties are most clearly displayed in the most centrally placed
cisternae. These are distended in the normal manner at one edge but not at the other,
Scale production in Prymnesium
377
the bunched, undistended, anteriorly directed margins being apparently oriented in
relation to the position of the entering flagella and haptonema base (Fig. 3). In addition, two or three of the most nearly central cisternae have local intercalary dilations
of the 'peculiar' sort (terminology of Parke, Manton & Clarke (1959)), common and
indeed characteristic of the Golgi body in many members of this group of flagellates,
more especially in the related genus Chrysockromulina (for a recent example see Manton,
1966). Such dilations can be filled with electron-opaque contents or may show only
traces of these against the inner faces of the distended membranes, as in this particular
fixation. Their functional significance is unknown but their presence, together with
the other marks of asymmetry mentioned above, is useful in the present context as a
means of recognizing the central cisternae as such, whether the entering flagellar
bases are included in the section or not (Fig. 4).
In addition to these features, a fairly constant attribute of certain Golgi cisternae
is the presence of included scales. These are undoubtedly in process of manufacture.
Several apparently mature scales can be seen in successive cisternae on the right-hand
side of both Figs. 3 and 4, while less fully formed ones can with care be made out in
cisternae somewhat closer to the Golgi centre in both specimens. Scale production is
however not encountered in any of the cisternae on the other side of the centre, i.e.
that nearest to the subtending endoplasmic reticulum.
Beyond the scale-containing vesicles in the direction away from the Golgi centre,
some large cavities, containing many contorted tubes, are present (Figs. 3 and 4,
right-hand side). These cavities also carry hairy diverticula, and scales can sometimes
be found among the tubes, though not in the micrographs reproduced. Both these
observations suggest that these cavities may represent former Golgi cisternae in some
late and perhaps final stage of development. Beyond and unrelated to them an apparently empty membrane-bounded space (Fig. 3, top right), designated previously as
the 'subcutaneous space' from lack of a better name (Manton & Leedale, 1963)
underlies the plasmalemma over almost the whole cell except the surfaces adjacent to
the flagellar appendages.
In this region there is one other feature of importance to which attention should
be directed in the present context. A large ' hairy' pit in the plasmalemma is commonly
present on one side of each flagellar base, comparable in size to the immersed portion
of the base itself. Examples are included in all the micrographs reproduced except
Fig. 1 (see especially Fig. 2). Figs. 3 and 4 only graze a pit incidentally, but Figs. 5 and
6 show clearly the alveolar pattern as seen in face view of the pit lining. Fig. 5 is
otherwise important as one of the rare examples of a longitudinal section passing
simultaneously through two pits, while Fig. 6 illustrates the longest pit so far traced
(almost 1 fi). Figs. 7 and 8 are two successive sections from a transverse series in which
two obliquely inserted pits, one on each side of the entering appendages, are present
(Fig. 8), though in such a case it is essential to be able to trace the innermost end of
the pit membranes (as in Fig. 7), since otherwise pits cannot be distinguished with
certainty from similarly patterned diverticula attached to Golgi cisternae and projecting towards the cell surface but not part of it.
There is no direct evidence regarding the functional significance of these pits. There
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is also little direct evidence regarding the mode of discharge of scales to the exterior,
except the fact that this almost certainly takes place close to the flagellar bases, since
access to the plasmalemma by scale-containing vesicles is virtually excluded on other
parts of the cell. This limitation nevertheless suggests, as a working hypothesis, that the
pits may mark the sites of scale liberation, inamanner to be discussed in the next section.
DISCUSSION
'Hairy', 'alveolar' or 'bristle-coated' vesicles and diverticula have been recorded
by different observers in so many different contexts in both plants and animals that a
unified function cannot at present be assigned to them. They have been cited in
association with protein uptake from the external medium into the oocytes of mosquitoes (Roth & Porter, 1964), attached to contractile vacuoles in the zoospores of
green algae (notably Stigeoclonium (Manton, 19646)), attached to the plasmalemma
near the place of discharge of contractile vacuoles in euglenoids (Leedale, Meeuse &
Pringsheim, 1965), attached to endoplasmic reticulum in certain animal cells (Novikoff
& Shin, 1964), apparently associated with digestive systems and the internal secretion
of enzymes in mammals and protozoa (Bruni & Porter, 1965; Tilney & Porter, 1965),
apparently associated with water control in the cells of the human placenta (Lister,
1963), to name only some of the more obvious recent papers. They are common in
many pigmentedflagellates,often attached to the Golgi cisternae as here and occasionally also attached to the plasmalemma, though rarely to the extent shown by the pits
in Prymnesium.
I do not propose even to suggest a function for them in this organism since only
biochemical experimentation could reveal this. As structural entities they can nevertheless perhaps be used as markers of differentiated membranes. Such membranes
are attached at all times to the scale-forming Golgi cisternae, as diverticula, and it is
by no means impossible that these might persist rather longer than the other, uncoated,
parts of the subtending cisternae. Scale liberation, it must be supposed, will in any
case involve fusion of some part of a mature scale-containing vesicle with the plasmalemma, after which the vesicle wall might either become incorporated into the plasmalemma or be resorbed into the cytoplasm. In either case a hairy pit could plausibly be
interpreted as the site either of initial fusion, or of the accumulation of remnants of
scale-containing vesicles after discharge, or as both. The great variability in size and
shape of the pits would fit in with such an explanation, though clearly more direct
evidence will be needed for it to become more than a working hypothesis.
Some rather more firmly based conclusions are possible with respect to a different
aspect of Golgi structure and function. Not only is there a regular sequence from
immature to mature scales, as cisternae recede from the Golgi centre in the direction
away from the subtending ER, but within each cisterna there is a regular orientation
of the developing scales such that these always face one way. As may be seen in Figs.
3 and 4, the inflexed rims of the internal scales face outwards with respect to the Golgi
centre and the flat part of the scale-base faces inwards. This must mean not only that
cisternae are maturing successively as they become pushed away from the centre,
Scale production in Prymnesium
379
presumably by the introduction of new cisternae behind them, but that within each
there is a functionally differentiated inner and outer face with respect to the Golgi
centre. This is expressed structurally by the very marked differences in surface
sculpturing and shape known to characterize the two faces of the scales that are being
formed in the cavity of the cisterna.
This type of evidence, giving new insight into functional aspects of the Golgi
complex which are difficult or impossible to observe directly, can be obtained from
many other pigmented flagellates, some of which are under active investigation from
this point of view. Prymnesium is a relatively simple case since the scales are all alike
and fairly small. Certain features, however, come out more clearly when conclusions
can be based on more than one example, and further discussion will therefore be
deferred until more comparative material is available. The facts as they stand nevertheless add significantly to the rapidly increasing body of knowledge regarding this
organism, and they can therefore appropriately be placed on record for their own sake.
REFERENCES
BRUNI, C. & PORTER, K. R. (1965). The fine structure of the parenchymal cell of the normal
rat liver. I. General observations. Am. J. Path. 46, 691-755.
LEEDALE, G. F., MEEUSE, B. J. D. & PRINGSHEIM, E. G. (1965). Structure and physiology of
Euglena spirogyra. I and II. Arch. Mikrobiol. 50, 68-102.
LISTER, U. M. (1963). Ultrastructure of the human mature placenta. I. The maternal surface.
J. Obstet. Gynaec. Br. Commomu. 70, 373-386.
MANTON, I. (1964a). The possible significance of some details of flagellar bases in plants.
Jl R. microsc. Soc. 82, 279-285.
MANTON, I. (19646). Observations on the fine structure of the zoospore and young germling of
Stigeoclonium. J. exp. Bot. 15, 399—411.
MANTON, I. (1964c). Observations with the electron microscope on the division cycle in the
flagellate Prymnesium parvum Carter. Jl R. microsc. Soc. 83, 317-325.
MANTON, I. (1964^). Further observations on the fine structure of the haptonema in Prymnesium parvum. Arch. Mikrobiol. 49, 315-330.
MANTON, I. (1966). Further observations on the fine structure of Chrysochromulina chiton with
special reference to the pyrenoid. J. Cell Sci. 1, 187-192.
MANTON, I. & LEEDALE, G. F. (1963). Observations on the fine structure of Prymnesium
parvum Carter. Arch. Mikrobiol. 45, 285-303.
NOVIKOFF, A. B. & SHIN, W. (1964). The endoplasmic reticulum in the Golgi zone and its
relations to microbodies, Golgi apparatus and autophagic vacuoles in rat liver cells. J.
Microscopic 3, 187—206.
M., MANTON, I. & CLARKE, B. (1959). Studies on marine flagellates. V. Morphology
and microanatomy of Chrysochromulina strobilus sp. nov. J. mar. biol. Ass. U.K. 38, 169-188.
REYNOLDS, S. (1963). The use of lead citrate at high pH as an electron-opaque stain in electron
microscopy. J. Cell Biol. 17, 208-211.
ROTH, T. F. & PORTER, K. R. (1964). Yolk protein uptake in the oocyte of the mosquito
Aedes aegypti L. J. Cell Biol. 20, 313-332.
TILNEY, L. G. & PORTER, K. R. (1965). Studies on microtubules in Heliozoa. I. The fine
structure of Actinosphaerium nucleofilum (Barrett), with particular reference to the axial rod
structure. Protoplasma 60, 317-344.
PARKE,
(Received 22 April 1966)
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Fig. 1. Prymnesium parvum. Two cells dried and shadow-cast, showing the two flagella
and short haptonema on each. Micrograph S 799/22, x 3000.
Fig. 2. Longitudinal section through aflagellumat its point of insertion into the cell,
showing a transverse section of a haptonema (top right) with traces of detached scales
in the ground outside the cell; inside the cell a mitochondrion and part of a plastid
(right), with part of the Golgi body showing cisternae in face view (bottom), some
with attached 'hairy' diverticula; a large 'hairy' pit attached to the plasmalemma
beside the flagellum. For further description see text. Micrograph C3856, x 50000.
Journal of Cell Science, Vol. i, No. 3
I. MANTON
(Facing p. 380)
Fig. 3. Longitudinal section through a flagellum cut in a plane at right angles to that
of Fig. 2, showing part of a haptonema base beside theflagellumand many loose scales
outside the cell (right). Within the cell the Golgi complex, cut longitudinally, occupies the space between the nucleus (below) and theflagellarbase; two superposed layers
of ER bound it at extreme left. For further description see text (p. 377). Micrograph C 4304, x 40000.
Journal of Cell Science, Vol. i, No. 3
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[. MANTON
Fig. 4. Oblique transverse section of the Golgi complex with the nucleus (left) and the
cell surface with part of a hairy pit cut by the edge of the picture (right), parts of two
chloroplasts, mitochondria and profiles of rough ER at top and bottom. For further
description see text (p. 377). Micrograph C5116, x 50000.
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I. MANTON
Fig. 5. Longitudinal section through a haptonema base and part of a flagellar base,
showing both lateral pits; at the bottom of the picture the Golgi body and part of the
nucleus, the Golgi cisternae seen in face view and with 'hairy' diverticula attached to
them (bottom left). Micrograph C5086, x 40000.
Fig. 6. An exceptionally deep pit, with the two flagella and haptonema of the organism
obliquely cut above. Micrograph C4989, x 40000.
Figs. 7, 8. Two successive sections from a transverse series through the flagellar and
haptonema base and associated structures. Two pits present, one on each side (arrows),
the alveolar marking on the terminal lining membranes visible at the deeper level
(Fig. 7). Other structures visible in Fig. 8 include parts of a chloroplast, mitochondria,
some fibres attached to the flagellar bases at a deeper level and tubes of rough-surfaced
ER. For further description of other aspects of this specimen see Manton (1964c).
Micrographs C4109 and C4115, x 40000.
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