J. Cell Sci. 14, 1-9 (1974)
Printed in Great Britain
ULTRASTRUCTURE OF AN UNUSUAL
CONTRACTILE VACUOLE IN SEVERAL
CHRYSOMONAD PHYTOFLAGELLATES
S. AARONSON AND U. BEHRENS
Biology Department, Queens College,
City University of New York,
Flushing, New York 11367, U.S.A.
SUMMARY
The infrastructure of the contractile vacuole of Ochromonas and Poterioochromonas species is
described. It appears to be a permanent structure in the anterior end of the phytoflagellate, consisting of a central cavity with a unit-membrane lining from which flattened saccules protrude
in all directions.
The contracted saccules are about 40 nm in diameter and are made up of a single trilammellar
unit membrane about 9 nm thick and continuous with the central cavity membrane. In their
contracted state the saccules contain transverse fibrils which may be involved in the contraction
of the saccules; these fibrils are no longer seen when the central cavity and the saccules swell.
The saccules seem to merge and become continuous with the central cavity during the swelling
of the contractile vacuole.
INTRODUCTION
Ochromonas and Poterioochromonas are closely related genera of phytofiagellates with
an unusual contractile vacuole which has not to our knowledge been described in detail
as here. In this paper we describe its ultrastrucrure.
METHODS
Ochromonas danica Pringsheim, Poterioochromonas (Ochromonas) malhamensis, P. sociabilh
(these 2 species are also known as variants of P. malhamensis) and Ochromonas sp. were obtained
from the Institute for Plant Physiology, the University, Gftttingen. O. danica was maintained on
the heterotrophic medium of Aaronson & Baker (1959); the other species were maintained on the
same medium supplemented with trypticase (0-5 %) and yeast autolysate (05 %). All cultures
were maintained at 25 °C in the light. Cells were removed from 7 to 8-day-old (mid to late-log)
cultures and harvested, fixed, and treated as described in Aaronson, Behrens, Orner & Haines
(1971). Light microscopy was done with a Nikon research microscope equipped with phase and
interference optics and light micrographs were taken with a Nikon F 35-mm camera body
attached to the microscope.
RESULTS
Living O. danica examined by light microscopy were found to contain one, rarely
two, contractile vacuoles located at the anterior end of the microorganism (Fig. 2).
These vacuoles expand (diastole) and contract (systole) with a frequency seemingly
dependent on the osmotic pressure of the medium (i.e. there is more vacuolar activity
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5. Aaronson and U. Behrens
in distilled water than in the growth medium which is almost isosmotic with the cells
(unpublished results of S. Benoff & S. Aaronson)). Attempts to study the energy
metabolism of the contractile vacuole with metabolic poisons proved to be inconclusive. Attempts to locate unusual structures in the contractile vacuole or a vacuolar
pore on the chrysomonad surface with light microscopy were unsuccessful.
Electron-microscopic examination of the several chrysomonad species revealed that
each contained a vacuolar structure made up of a central cavity or sac from which
several seemingly tubular evaginations projected out into the cytoplasm (Fig. 3). No
differences in the contractile vacuole of the several species were noted, although in
O. danica a plane of section through this organelle was encountered less frequently
than in the other species. Study of hundreds of chrysomonads revealed that these
seemingly tubular projections from the contractile vacuole rarely appeared as circles,
which would be expected if they were tubular structures. The evaginations most frequently appeared as flattened structures with rounded ends, suggesting cross-sections
of a flattened saccule. From this we deduce that the contractile vacuole in systole
appears as an open sac-like vacuole from which extend a variable number of flattened
saccules, giving the vacuole a pleated appearance somewhat like that of an ice pack.
As these saccules are most frequently seen as fairly long parallel-sided tubes with
rounded ends (Figs. 3-5) in cross-section, we assume that they have relatively small
openings into the central vacuolar space. A diagrammatic version of how we think the
organelle may appear in 3 dimensions is presented in Fig. 1; the figure shows a view
into the centre of the vacuole through a partly cut away wall, with the oval holes representing the openings of the saccules into the main cavity. The flattened saccules extend
through the anterior end of the organism and may occasionally be seen to extend almost
halfway down its length (Fig. 4). The flattened saccules, about 40 nm in diameter, are
made up of a single trilamellar unit membrane about 9 nm thick which is continuous
with the same unit membrane in the central vacuolar space (Fig. 5). Occasionally, tiny
granules appear to be attached to the cytoplasmic side of the membrane (Fig. 6). These
granules were not found consistently. The flattened saccules appear to be held in
position by numerous fine fibrils which extend across them (Figs. 3,5,6,10,11). These
fine fibrils are no longer seen when the saccules fill with fluid and merge with the
central vacuolar space to form the contractile vacuole in diastole. In diastole, the contractile vacuole seems to enlarge enormously, presumably at the expense of the flattened saccules which are no longer seen or appear to be reduced in number (Fig. 7),
and eventually the vacuole presses up against the plasma membrane (Figs. 5, 7).
While we have no light-microscopic evidence for a vacuolar pore to the outside, at the
ultrastructural level we find a pseudopodial-like projection containing a single flattened
saccule (Fig. 8) which is continuous with the contractile vacuole and leads toward the
cell exterior, but we have not seen an unambiguous opening (Fig. 8). A possible pore
opening associated with the appropriate vacuole region but lacking a projection was
seen (Fig. 9). In the light microscope the swelling contractile vacuole is seen clearly,
but it disappears on bursting to the exterior at diastole. At the ultrastructural level we
frequently see a depression in the cell surface (Figs. 5, 7) which may be the appearance
just after the vacuole has emptied and the saccules are reforming. That the flattened
Ultrastructure of contractile vacuole
3
saccules may fuse with each other and form loops and other combinations may be
seen in Figs. 1 o and 11. There is some ultrastructural evidence that at some part(s) of
the contractile vacuole (perhaps the top and bottom) several saccules anastomose or
converge (Fig. 12; see also top and bottom of Fig. 1).
Fig. 1. Highly diagrammatic version of the 3-dimensional appearance of the Ochromonas contractile vacuole. A view into the central vacuolar cavity through a cut. The
vertical tubes represent saccules, the oval openings are holes from saccules into the
central cavity.
DISCUSSION
The contractile vacuole of the several chrysomonads studied, like that of many other
flagellate protozoa, is located anteriorly and in close proximity to the Golgi and the
flagella. No functional or structural relationship between the contractile vacuole and
the aforementioned organelles was apparent in these Ochromonas or Poterioochromonas
species.
Hibberd (1970) has described a similar structure in Ochromonas tuberculatus sp.nov.
He was unable to make out the probable 3-dimensional structure of the contractile
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S. Aaronson and U. Behrens
vacuole but he did see the fine fibrils in the saccules and the concavity of the cell surface in the region overlying the contractile vacuole in diastole.
Tsekos & Schnepf (1972) described a very similar contractile vacuole in the closely
related chrysomonad, Poterioochromonas stipitata. They described a flattened saccule
with perpendicular fibrils inside, very much like those seen here. They did not describe
the vacuolar structure in detail but reported particles associated with the outer surface
of the flattened sac which they suggest might be associated with energy metabolism.
We have seen these particles but were unable to find them consistently associated with
the contractile vacuole's saccules. They reported a membrane thickness of 7-5 nm
which is similar to the value of 9 nm reported here.
Among protozoan contractile vacuoles that of the chrysomonads bears some resemblance to the pusule of dinoflagellates although the pusule has 2 unit membranes
and empties directly into aflagellarcanal (Leadbeater & Dodge, 1966; Mignot, 1970;
Schnepf & Deichgraber, 1972; see Dodge, 1972, for a review). The details of Ochromonas and Poterioochromonas contractile vacuole function, like that of the dinoflagellate
pusule, remain to be determined.
Aided by grants to S.A.: GB 20825 from the National Science Foundation and in part by
grant N I H 5-S05-RR-07064 from the National Institutes of Health to Queens College. We
also wish to express our thanks to Dr A. T . Soldo for valuable discussion and to Martin
Lynn for drawing Fig. 1.
REFERENCES
AARONSON, S. & BAKER, H. (1959). A comparative biochemical study of two species of Ochromonas. J. Protozool. 6, 282-284.
AARONSON, S., BEHRENS, U., ORNER, R. & HAINES, T . H. (1971). infrastructure of intracellular
vesicles, membranes, and myelin figures produced by Ochromonas danica. J. Ultrastruct. Res.
35, 418-430DODGE, J. D. (1972). The ultrastructure of the dinoflageUate pusule: a unique osmoregulatory
organelle. Protoplasma 75, 285-302.
HIBBERD, D. J. (1970). Observations on the cytology and ultrastructure of Ochromonas tuberculatus sp. nov. (Chrysophyceae), with special reference to the discobolocysts. Br. phycol. J. 5,
"9-143LEADBEATER, B. & DODGE, J. D. (1966). The fine structure of Woloszynskia micra sp. a new
marine dinoflagellate. Br. phycol. Bull. 3, 1—17.
MIGNOT, J. P. (1970). Remarques sur le developpement du reticulum endoplasmique et du
systeme vacuolaire chez les gymnodiniens. Protistologica 6, 267-281.
SCHNEPF, E. & DEICHGRABER, G. (1972). Uber den Feinbau von Theka, Pusule und GolgiApparat bei dem Dinoflagellaten Gymnodinium spec. Protoplasma 74, 411-425.
TSEKOS, I. & SCHNEPF, E. (1972). Partikel an der Membran der Kontraktilen Vacuole von
Poterioochromonas stipitata. Naturwissenschaften 59, 272-273.
(Received 26 April 1973)
ABBREVIATIONS ON PLATES
c chloroplast
cv contractile vacuole
/
fibrils
m mitochondrion
n nucleus
pm plasma membrane
s saccule
Ultrastnicture of contractile vacuole
Figs. 2-4. For legends see p. 6.
S. Aaronson and U. Behrens
Fig. 2. O. danica. Light micrograph showing round contractile vacuole during
diastole (arrow), x 2000.
Fig. 3. P. sodabilis showing contractile vacuole and connecting and seemingly unconnected saccules. x S3 200.
Fig. 4. P. sodabilis. Saccules (arrows) almost halfway down length of the organism,
x 28500.
Fig. 5. P. malhamensis. Contractile vacuole up against plasma membrane, showing
saccules and saccule membrane continuous with vacuolar membrane, x 71 800.
Fig. 6. Enlargement of a saccule opening into vacuole with fine transverse fibrils and
tiny granules (arrows) on cytoplasmic side of trilamellar membrane, x 123000.
Fig. 7. P. malhamensis. Contractile vacuole membrane located close to plasma membrane. Note indentation in both membranes. Presumably a stage just after expulsion
of vacuole contents, x 31 500.
Ultrastructure of contractile vacuole
J
S. Aaronson and U. Behrens
Fig. 8. P. sociabilii. Pseudopodial projection with single saccule continuous with
contractile vacuole. x 66 500.
Fig. 9. O. danica. Contractile vacuole and associated saccules. Possible saccule or
pore to cell exterior (arrow), x 39000.
Fig. 10. P. sociabilis. Loop formed by a saccuJe and continuous with arm of contractile
vacuole. x 78000.
Fig. 11. P. sociabilis. Circular saccule. x 102000.
Fig. 12. P. malhamensis. Anastomosis of saccules presumably at top or bottom of contractile vacuole. x 79800.
Ultrastructure of contractile vacuole