The formation of siliceous scales by Raphidiophrys ambigua (Protista,
Centroheliozoa)
DAVID J. PATTERSON
Department of Zoology, Univeisity of Bristol, Bristol BSS 1UG, England
and MONIKA DURRSCHMIDT
Institut fur Pflanzenokologie, Universitat Giessen, Heinrich Buff Ring 38, D 6300 Giessen, IV. Germany
Summary
Ultrastructural aspects of the formation of siliceous
scales by the protozoon Raphidiophrys ambigua
are described. Cells in culture become scale-free in
silicon-impoverished medium. Scales may be seen
adhering to the cell surface within four to six hours
of the addition of silicon to the medium. Silica scale
formation occurs within deposition vesicles (SDVs)
near the periphery of the cell. Many scales are
formed at the same time; each may be in a different
phase of scale formation. The source of the SDV
membrane is not known. Silicification proceeds
centrifugally from two pattern centres. The first
parts of the forming scale are two opposed sterna,
which establish the longitudinal axis of the scale.
One sternum develops from each pattern centre.
The scale develops outwards by formation of fairly
evenly spaced lateral ribs from each central sternum. Distally, the ribs branch to form a reticulate
pattern. A thin continuous sheet of silicon is added
to the periphery of the scale, curving inwards to
form a rim. The development of this pattern may be
described in terms of a small number of morphogenetic processes.
Introduction
see Durrschmidt, 1985; Croome, 1987).
In contrast to diatoms, centroheliozoa can be cultured
readily in silicon-free (i.e. impoverished) media (Patterson & Durrschmidt, 1986), under which conditions scaleless cells eventually predominate. These will re-form
scales when silicon is added. This paper is an account of
ultrastructural aspects of scale formation induced in this
way in the acanthocystid Raphidiophrys ambigua. The
general ultrastructure of this species has been described
elsewhere (Durrschmidt & Patterson, 1987). Other aspects of the cell biology of similar (centrohelid) heliozoa
are given by Bardele (1975, 1977a,b), Davidson (1976),
Febvre-Chevalier & Febvre (1984), Rieder (1979) and
Tilney (1971).
Silicon is used by a wide variety of phylogenetically
remote protists to form walls, tests or skeletal structures
(Simpson & Volcani, 1981; Leadbeater & Riding, 1986).
Most of the information about mechanisms of protist
silicification relates to diatoms (Schmid & Schulz, 1979;
Schmid et al. 1981; Volcani, 1981; Schmid, 1984;
Crawford & Schmid, 1986; Pickett-Heapsei al. 1988, for
reviews). Some additional information is available for
chrysophytes (Schnepf & Deichgraber, 1969; Mignot &
Brugerolle, 1982; Brugerolle & Bricheux, 1984; Leadbeater, 1984; Preisig, 1986), choanoflagellates (Leadbeater, 1984, 1985, 1986), testate amoebae (Netzel, 1972;
Bovee, 1981; Harrison et al. 1981; Anderson, 1987),
filose amoebae (Patterson, 1985), radiolaria (Anderson,
1983, 1986), heliozoa (Patterson & Thompson, 1981;
Patterson & Durrschmidt, 1986) and sponges (Garroneei
al. 1981).
Siliceous structures are formed by at least some
members of each type of heliozoon (Patterson & Durrschmidt, 1986), and are formed by all members of the
centrohelid family Acanthocystidae (see Lee et al. 1985,
for a recent general account of this group). The siliceous
artefacts are used extensively in taxonomic studies (e.g.
Journal of Cell Science 91, 33-39 (1988)
Printed in Great Britain © The Company of Biologists Limited 1988
Key words: Protozoa, Heliozoa, silica, morphogenesis.
Materials and methods
Raphidiophrys ambigua was obtained as stock 1568/1 from the
Culture Centre for Algae and Protozoa (Cambridge, UK). Cells
were maintained in a mineral water (Evian) containing silicon at
1-35 mgml" 1 . Assuming that the silicon formed a saturated
solution, the concentration of free silicon is likely to be in the
vicinity of 5 mM (Alexander et al. 1954). Silicon-impoverished
Chalkley's medium (Chalkley, 1930) was made up using fresh
33
Figs 1-4. These and all subsequent figures are of R. ambigua.
Fig. 1. Light micrograph of a normal cell surrounded by scales, the long filamentous structures are axopodia. X 1000.
Fig. 2. Light micrograph of a cell from silicon-impoverished medium, there are no scales. X1000.
Fig. 3. Light micrograph of a living cell 4h after re-silicification. The arrows indicate developing scales. X 1000.
Fig. 4. Scanning electron micrograph showing the boat-shaped scales. X4500.
deionized water and Analar grade chemicals. All solutions,
cultures etc. were maintained in plastic containers that had not
been tissue treated (the process can add large amounts of
silicon). The heliozoa were fed every 2 days with Polytomella
papillata (CCAP, 63/2c) washed with silicon-free Chalkley's
medium.
Cells were fixed for 30min at 4°C by the rapid addition of
2-5 % glutaraldehyde, 1 % osmium tetroxide and 5 mM-magnesium chloride in 50mM-sodium cacodylate buffer (pH7-4).
Cells were washed and dehydrated to 50% ethanol and then
stained for 2h in a saturated solution of uranyl acetate in 50%
ethanol. The cells were dehydrated and embedded in a thin film
of Araldite, for subsequent sectioning of individual cells (Patterson, 1985).
Cleaned whole mounts of isolated scales were prepared by
digesting cells in dilute (25%) nitric acid for 30min, washing
several times in distilled water before mounting on electronmicroscope grids.
Results
Normal R. ambigua cells have a rounded body from
which extend a small number of stiff radiating axopodia
that are used in food capture (Fig. 1). Around the cell
body lies a periplast of loosely adhering boat-shaped
scales (Figs 1,4). The scales are constructed as a thin,
flat mesh of siliceous material, with the edges curved over
to form a rim (Figs 4, 5, 21). Most scales measure from
1-5/zm X 4 Jim to 2-5/xm X 8/zm (Fig. 6). The terms by
which we refer to components of the scale are indicated in
the legend to Fig. 5.
Cells cultured in silicon-impoverished or silicon-free
conditions lose their scales over a period of several weeks
(Fig. 2). The cells continue to divide, and scale loss may
result either from dissolution or progressive reduction in
number through redistribution to daughter cells. Scales
begin to appear at the surface of previously scale-less cells
within 4—6 h of the re-addition of silicon-rich Evian water
(Fig. 3).
The ultrastructure of scale-less cells from silicon34
D. jf. Patterson and M. Durrschmidt
Fig. 5. Line drawing to illustrate most major parts of the
scale. 1, pattern centre; 2, isthmus; 3, sternum; 4, ribs;
5, mesh; 6, rim.
impoverished conditions is similar to that of normal cells.
A centroplast lies at the centre of the cell and gives rise to
the microtubular axonemes that support the axopodia.
Most organelles are arranged in concentric zones around
the centroplast, with the single nucleus lying eccentrically. Differences compared with normal cells (Fig. 8)
are: a lack of any mature or forming scales or silicon
14
12
10
Scale breadth (jtm)
Fig. 6. Lengths and breadths of scales plotted against each
other for scales from normal cells (after Diirrschmidt &
Patterson, 1987).
deposition vesicles (SDVs); a relatively large amount of
lipid; and a reduced lacunar system. With the exception
of reappearing SDVs, these features may be seen in
Fig. 7, which shows a cell within hours of the readdition
of silicon.
SDVs are evident in sections of cells fixed 4h after
being returned to Evian water. They appear immediately
below and apparently attached to the plasma membrane,
remaining in this location until the scale is fully formed.
The first identifiable SDVs are relatively small (Figs 9,
10). No cytoskeletal structures that might be imposing
form on developing scales have been observed consistently.
The first discernible siliceous structure is a central
fibre or sternum (Figs 9, 15). Thereafter, transverse
sections show more expansive arrays of silicon as discontinuous (granular) extensions from the sternum (Figs
10-13). Silicification is always heaviest in the vicinity of
the sternum and weakest towards the periphery of the
scale (Figs 9-13).
Small vesicles are often seen in association with the
SDVs, prior to the appearance of silica or during earlier
stages of scale development. The vesicles are especially
evident in the region of the sternum (Figs 9, 11, 12). No
inclusions have been observed in these vesicles.
One of the last events in scale development is the
formation of the curved marginal rim. Scales with fully
formed rims often occur in SDVs that lie away from the
plasma membrane (Fig. 13). They must later return to
the periphery of the cell so that the scale may be
exocytosed (Fig. 14).
Preparations of acid-cleaned whole scales help to
clarify the sequence in which development occurs. The
least developed siliceous artefacts (presumed to be of
scales early in their development) consist of the thickened
central sterna with a few poorly developed lateral ribs
(Fig. 15). The mid-point of the sternal elements is
narrow, heaviest silicification occurring in two regions,
one on either side of the midpoint. These 'pattern centres'
are separated by an 'isthmus' (Figs 15, 16). The lateral
ribs arise at fairly evenly spaced intervals along opposing
sides of each sternum (Figs 15—21). The ribs first extend
as independent siliceous threads (Fig. 15), but then
branch and anastomose to form a mesh (Figs 17-18).
Unbranched ribs are longest near the isthmus, and
shortest near the tip of the scale. A thin continuous sheet
of silicon is added at the margin of the mesh (Fig. 19),
eventually curving up to form the marginal rim (Figs 20,
21). Small drop-shaped fragments of silica were associated with some siliceous scales (Figs 16, 17). The pattern
in the scales develops from the central region outwards,
and in whole mounts all peripheral regions are always at
the same stage of development.
During this work, we were able to observe a number of
deformed (teratological) scales (e.g. see Figs 22-28).
Some had a single pattern centre with (Figs 23, 24) or
without (Fig. 22) a sternum. Other scales had more than
two sterna (Figs 25, 27, 28), each arising from a separate
pattern centre. In all cases ribs had formed radially from
the pattern centre and/or sterna, with the lattice and rim
extensions. A few scales that lacked the mesh in the midregions (Figs 26, 27) had pattern centres that were very
distant from each other.
Discussion
A great diversity of, mostly protistan, organisms fabricate
siliceous artefacts. Most information about biological
silicification relates to diatoms (see Schmid et al. 1981),
and consequently few general concepts have emerged
about biogenic silica deposition. Perhaps the only two are
that amorphous silica forms within a membrane-bound
silicon deposition vesicle (SDV), and upon deposition
the artefact has a predictable form. There are two
phenomena to be discussed, that of silicification (the
deposition of solid silica) and that of pattern formation
(the factors controlling the form of the deposited artefact).
Silicification occurs within an SDV. The source of the
SDV in Raphidiophrys is unresolved. Schmid (1984)
proposed that SDVs in diatoms may arise from dictyosomes, although there is no evidence that would support a
similar interpretation in other protists (Leadbeater &
Riding, 1986). As in diatoms and chrysophytes (Schmid
& Schulz, 1979; Brugerolle & Bricheux, 1984; Li &
Volcani, 1985; Crawford & Schmid, 1986), small vesicles
were observed around the SDVs of R. ambigua. No
Silicification of heliozoan scales
35
mm*
8
Figs 7-14. Transmission electron micrographs of sectioned material showing development of scales.
Fig. 7. Thin section of a cell from silicon-impoverished medium, 4h after adding silicon medium. Arrows show SDVs;
c, centroplast; n, nucleus;/, food vacuole; /, lipid. X4700.
Fig. 8. A cell 24 h after re-silicification; n, nucleus. X6600.
Fig. 9. An SDV at a very early stage of development containing only a sternum. Note adjacent vesicle (arrow). X78 000.
Figs 10, 11. Developing scale with the thicker central sternum (arrowhead) and differing degrees of lateral development. Note
adjacent vesicles (arrows). Fig. 10, X70000; Fig. 11, X71000.
Fig. 12. Well-developed scale lying under the plasma membrane, note vesicles (aiTows). X6SOO0.
Fig. 13. Scale in final form, still within the cell and the SDV is no longer attached to the plasma membrane. X48000.
Fig. 14. Exocytosed scale lying on the surface of the cell. X60000.
36
D. jf. Patterson and M. Diirrschmidt
15
16
17
18
19
20
Figs 15-21. Whole mounts of scales in various stages of development, arranged roughly in what is believed to be the
developmental sequence (see the text), x 10000.
28
Figs 22-28. Whole mounts of teratological scales, showing some of the abnormalities that may be encountered. See the text for
details. X 10000.
inclusions were seen in the vesicles and we believe that
they simply contribute membrane to the expanding
SDV.
Within the SDV, the first structures to be visibly
silicified are the pattern centres (a term taken from the
diatom literature: Schmid el al. 1981; Mann, 1984), one
at the base of each sternum. These oldest regions are the
most heavily silicified of mature scales. We suggest that
this is most simply explained by assuming that there is a
continuous accretion of silica to all preformed structures
within the SDVs, so that thickness of silicon in any region
is merely a function of the age of that region.
As in filose amoebae, diatoms, radiolaria and some
chrysophytes (sensu lato) (Schmid, 1980; Mann, 1984;
Silicification of heliozoan scales
37
Patterson, 1985; Anderson, 1986; Pickett-Heaps et al.
1988), silica may be deposited as thin threads in forming
Raphidiophrys scales. This suggests that there may be
some constraints as to how silicification may be achieved,
but it is also noticeable that siliceous deposits are formed
differently in different taxa. Some, for example, deposit
silica around a central organic core (Garrone et al. 1981;
Leadbeater, 1984, 1986; Anderson, 1987). Actinophryid
heliozoa assemble a sintered matrix that accretes additional silica to become amorphous (Patterson &
Diirrschmidt, 1986). We infer from this variety that the
pattern of deposition is not a result of the molecular
organization of silica (self-assembly), but can be imposed
by the cell. The means by which this is achieved may be
referred to as pattern formation or morphogenesis.
Some aspects (pattern centres, sterna, mesh) of the
pattern observed in scales of R. ambigua resemble early
developmental stages of diatom frustules (Mann, 1984).
Diatoms are believed to be related to chrysophytes
(Round & Crawford, 1981) and phylogenetically remote
from centroheliozoa (Smith & Patterson, 1986).
Although the similarities of pattern are unlikely to be
homologous, they are sufficiently great for us to discuss
R. ambigua in relation to diatoms. We have adopted a
changed use of terms for centroheliozoan scales to
facilitate that discussion (cf. Diirrschmidt & Patterson,
1987).
As in diatoms, the pattern of the centroheliozoan
artefact develops progressively as silicification occurs (cf.
actinophryid heliozoa in which the form of the finished
artefact appears to be established before silicification
begins). However, unlike diatoms, many siliceous artefacts may be produced in centroheliozoa at any one time,
and these may be at differing stages of development. The
mechanisms that control the pattern are not therefore
expressed throughout the entire cell, but at the level of
individual SDVs. However, as all margins of any scale
seem to be at the same stage of development at any time,
we conclude that only one facet of morphogenesis can
occur within an SDV at any one time.
Normally the membrane of the SDV follows the
contour of the artefact. A simple question arises: 'is the
SDV shaped by the developing scale, or does the SDV
impose shape on the scales?' Both mechanisms probably
operate. Microtubular and/or microfilamentous structures appear around the SDVs of diatoms, chrysophytes
and choanoflagellates (Schmid, 1980; Pickett-Heaps &
Kowalski, 1981; Brugerolle & Bricheux, 1984; Leadbeater, 1984; Li & Volcani, 1985; Leadbeater & Riding,
1986; Pickett-Heaps et al. 1988). In such organisms,
damage to microtubules, or the distortion of the cell by
osmotic shock, will lead to the production of deformed
siliceous structures (Schmid, 1979, 1980, 1982; Leadbeater, 1984). The shape of the siliceous artefact evidently can be determined in whole or in part by elements
in the cytoplasm. Alternatively, organic matter can be
used as a template for silicification (Heckey et al. 1973;
Leadbeater, 1984; Simkiss, 1986). No cytoskeletal structures have been noted in the vicinity of the forming scales
of centroheliozoa. Some evidence for the existence of an
organic component in centroheliozoan scales that may act
38
D. J. Patterson and M. Diirrschmidt
as a template has been presented, but this requires
verification (Patterson & Diirrschmidt, 1986). We believe
that the tight association between the SDV and the
plasma membrane may help to control the shape of the
SDV and therefore that of the forming scale.
The pattern develops centrifugally from the pattern
centres. Normal scales have two pattern centres separated
by an isthmus. As in pennate diatoms, a sternum grows
from each pattern centre, typically in opposite directions.
The pattern develops around this axis, with axial growth
occurring more rapidly than outward (lateral) growth to
produce long boat-like scales. A change in the relative
rates of growth in the axial and centrifugal directions
ought to produce broad leaf-like scales or narrow scales.
Such scales do occur in this and related species (Diirrschmidt & Patterson, 1987), indicating that this is one
morphogenetic variable that has been adjusted during
evolution to produce some of the variety in scale form.
No scales have been found without a pattern centre, so
this is regarded as a necessary part of scale development.
Sterna are usually the major expression of axial growth,
but they may be absent. They appear not to be a
necessary stage in scale formation.
The outward (radial) growth has three components:
ribs, mesh and rim. The rib and mesh combination is
very similar to that expressed during early frustule
development in diatoms, although on a somewhat smaller
scale (Mann, 1984). Ribs, mesh and margin all appear to
be obligate components of the outward development of
the scale. They occur around the pattern centres and/or
ribs irrespective of their number and location. The shape
of the scale can therefore be predicted from the number
of pattern centres, the distance between them, and the
number of sterna. A similar dependence of the shape of
the final structure on early stages of formation is encountered in diatoms, where distortion of the pattern centre
leads to deformation of the frustule (Schmid, 1980).
From these observations we conclude that the pattern
centres, sterna, ribs, mesh and rim are produced as five
morphogenetic events separated in time. The formation
of ribs, mesh and rim (being inseparable) most probably
represent different phases of one process, but the number
of pattern centres and the formation of sterna appear to
be independently controlled.
This work was carried out during tenure of a Deutsche
Forschungsgemeinschaft (DFG) postdoctoral fellowship by
M.D. at the University of Bristol. M.D. thanks the head and
staff of the Department of Zoology for providing facilities and
for their assistance. We especially thank K. Williams for his able
technical support.
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(Received 12 January 1987 - Accepted, in revised fonn, 25 May 1988)
Silicification of heliozoan scales
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