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B i d Rm (1980). 55. pp 1-46
With 5 plates
Printed in Great Britain
CONTRACTILE VACUOLES AND ASSOCIATED
STRUCTURES : THEIR ORGANIZATION AND FUNCTION
BY D . J . PATTERSON
Department of Zoology. University of Bristol. Bristol BS8 I UG. England
(Received 29 August 1979)
I . Introduction
I1 Organization
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CONTENTS
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(I) Morphology
(a) Terminology and general comments
(b) Types of morphological organization
( c ) Artefacts
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(2) Behaviour
(a) General comments and terminology
(b) Typesof behaviour
(c) Observation of contractile vacuoles
(3) Systematic survey of the distribution and organization
of contractile vacuole complexes
(4) Towards a new concept of contractile vacuoles .
I11 Factors affecting activity of contractile vacuole complexes .
IV Suggested functions of contractile vacuole complexes .
(I) Osmoregulation
(2) Feeding
(3) Ion regulation
(4) Excretion of waste metabolites
( 5 ) Respiration and circulation
(6) Reproduction
(7) Enzyme outlet
(8) Excystment
(9) Lorica formation
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(10) Mucus secretion
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V The production of vacuolar fluid and its control
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( I ) The mechanism of fluid production
(a)Water transport
(b) The osmoticmechanism
( c ) Phase separation
(d) Structural segregation
(2) Control of fluid production
VI . Vacuolar contractility
(I) The case for contractility
(2) Control of the contractile process
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VII . Ontogeny and phylogeny of contractile vacuole complexes
VIII Summary
IX Acknowledgements
X . References
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1980 Cambridge Philosophical Society
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2
D. J. PATTERSON
I. INTRODUCTION
Contractile vacuoles are subcellular organelles which are defined by their behaviour
of filling slowly with fluid, and periodically expelling their contents from the cell.
The frequency of expulsion varies from once every few seconds to twice per hour, or
longer (Kitching, 1938b). The frequency of expulsion can be modified by changes in
external factors, particularly the osmotic pressure of the bathing medium.
Contractile vacuoles form one part of a more extensive system which can now be
more adequately described with the help of the electron microscope. I propose to
follow de Saedeleer & Wolff ( 1 9 3 1 ~ and
)
call this entire system the ‘contractile
vacuole complex’.
Contractile vacuoles are generally considered to be protozoan organelles, being
usually present in fresh-water representatives of the ciliates, flagellates and amoebae,
and are sometimes in marine and parasitic representatives of these groups. They are
also found in many unicellular algae and at certain stages in the life cycles of some other
algae and fungi.
The first observation of contractile vacuoles has been attributed to Joblot (Woodruff, 1937; Wichterman, 1953; Organ, Bovee & Jahn, 1972). However, a critical
reading of the relevant text (Joblot, 1718)fails to substantiate this. The first clearly
recorded observations of contractile vacuoles appear to be those of Spallanzani (1776).
Much of the early research on contractile vacuoles was directed toward answering
such simple questions as: Is there a membrane around the vacuole?, Where does the
fluid come from?, Where does the fluid go to?, etc. These questions had been satisfactorily answered by the earlier part of this century (Jennings, 1904; Taylor, I923 ;
Howland, 1927b; Howland & Pollack, 1 9 2 7 ~ (i.e.
) there is a membrane, the fluid
is derived from the cytoplasm and is expelled from the cell). From the end of the
last century the evidence had suggested that the contractile vacuole has a r61e to
play in protozoan osmoregulation (Rossbach, 1872; Hartog, 1888; Degen, I905 ;
Herfs, 1922). This provided a basis for subsequent illuminating experimental studies,
in which Kitching played a major r61e (Kitching, 1934, 1936, 1938u, I939U, b, c,
1948a, b, c, 1951, 195zb, 1954b, c; Kitching & Padfield, 1960), and these have confirmed that the organelle is involved in osmoregulation.
The last two decades have produced major developments in our understanding of
the morphological organization of the organelles through electron-microscopical
studies, with Rudzinska (1958) and Schneider (1960) setting the early standards,
and McKanna (1972, 1973b, 1974, 1976) the later ones. Ion-physiological studies
have also influenced our concepts of the functions of contractile vacuoles (Dunham
& Child, 1961; Stoner & Dunham, 1970; Dunham & Kropp, 1973; Prusch, 1977).
The literature on contractile vacuoles has been reviewed several times in the past
;
1938b, I952a, 1954u, 1956, 1967; Weatherby, 1941).
(Lloyd, 1 9 2 8 ~Kitching,
11. ORGANIZATION
This Section discusses the information on the organization of contractile vacuole
complexes as inferred from their structure and behaviour.
As the discussion of the biology of contractile vacuoles is hampered by the absence
3
Contractile vacuoles
of a consistent and generally accepted terminology, some recommendations made by
Patterson & Sleigh (1976) are repeated and elaborated below. Text-figs I and 2
illustrate some of the more common patterns of structural organization and behaviour
of contractile vacuole complexes.
(I)
Morphology
(a) Terminology and general comments
One part of the contractile vacuole complex may be seen to fill slowly with fluid and
periodically expel this fluid from the cell. This is the contractile vacuole itself, and is
frequently the only part of the complex visible with the light microscope. Recently
an alternative term, ‘the water expulsion vesicle ’, has been introduced on the grounds
that the contractile nature of the vacuole was unproven (Jahn, Rinaldi & Wigg, 1964;
Wigg, Bovee & Jahn, 1967).
When viewed with an electron microscope, the contractile vacuole is usually seen
to be surrounded by a system of membranous vesicles and/or tubules (the spongiome).
There may also be associated canals and a pore to the exterior. Together these form
the contractile vacuole complex.
Alternative names for the contractile vacuole complex, such as the nephridial
apparatus or the excretory apparatus (Nassonov, 1924; Gelei, 1925, 1928, 1933,
1938; Organ, Bovee & Jahn 1968a; Organ et al., 19683;Hausmann & Allen, 1977)
misleadingly imply a functional homology with metazoan kidneys. The analogy with
the passage of fluids from the body is clear but there is no reason to suppose any
further similarity.
The majority of contractile vacuoles are surrounded by a system of fine (20-50 nm)
membranous tubules or vesicles. This was first described by Gelei after impregnation with heavy metals and referred to as ‘ Spongyom’ (Gelei, 1928)and is now called
the spongiome. The addition of words such as ‘tubular’, ‘vesicular’, ‘decorated’ etc.
may be used to distinguish between the different types of spongiome seen under the
electron microscope (Text-fig. I, Section 11, I, b). Some components of the spongiome in certain species may be seen with the light microscope. For example, in many
amoebae the spongiome is made up of submicroscopicvesicles which enlarge to become
visible with the light microscope. These larger or contributory vesicles, fuse with, or
fuse together to form, the contractile vacuole. Furthermore, in a number of larger
ciliates the tubules of the spongiome connect with a system of large collecting canals
which feed into the contractile vacuole. These are particularly obvious in the peniculine ciliates such as Paramecium (Section 11, I , b). These and many other ciliates
have distensible regions of the spongiome, the ampullae, which dilate as they accumulate fluid derived either from the contractile vacuole or from other parts of the
spongiome.
In all ciliates so far investigated, the contractile vacuole discharges its contents
through a permanent pore. This is an indentation of the plasma membrane which is
supported by two sets of microtubules. Helical pore-microtubules wind around the
pore and appear to be firmly attached to the plasma membrane. Bands of radial
pore-microtubules extend from these and pass over the poreward surface of the
1-2
4
D . J . PATTERSON
D
c
Text-fig. I . Four types of morphological organization of contractile vacuole complexes. (A)
The contractile vacuole has a permanent pore (CVP) which is supported by helically wound
microtubules (HPMT) and from which bands of microtubules (RPMT) extend radially over
the poreward hemisphere of the contractile vacuole. Smooth tubular spongiome (STS) connects with the contractile vacuole. This type of organization is found primarily in kinetofragminophoran ciliates (Section 11. 3). (B) T h e contractile vacuole has a pore similar to that of
type A. The spongiome is elaborate, containing decorated tubules (DTS) arranged in the
fascicles of the orthotubular system (OTS) as well as smooth tubules (STS) which join the
decorated elements to collecting canals (CLC). The collecting canals are supported by extensions of the radial pore microtubules and have a distensible region near the contractile vacuole,
the ampulla (AMP). This is separated from the vacuole by a short indistensible connecting
canal (CNC). T h e insert shows the collecting canal as seen in cross section with supporting
microtubules and associations with the smooth tubular spongiome. This type of organization
is typical of the peniculine ciliates, while a similar type of organization, but lacking the
well-developed collecting canals, is typical of many other Oligohymenophora. (C) The
contractile vacuole is surrounded by a spongiome of submicroscopic vesicles (VS) and beyond
that by a layer of mitochondria (M). The vesicles and the contractile vacuole membrane bear
irregular coats on their cytoplasmic surfaces. This type of Organization is typically found in
amoebae. (D) The contractile vacuole is surrounded by a spongiome comprised of irregular
vesicles and tubules (TVS). This type of spongiome frequently contains some regular, 100
nm, coated vesicles (CTDV). This type of organization is typical of many flagellates and
smaller amoebae. For references see relevant groups in Section 11, 3.
vacuole and may continue to run along the collecting canals (McKanna, 1973b;
Hausmann & Allen, 1977; Allen 1978a, b) (PIS I a, b, z a , b). The pore is normally
sealed by a diaphragm made up of two membranes (the invaginated plasma membrane
and the contractile vacuole membrane) and a thin layer of cytoplasm (McKanna,
Contractile vacuoles
5
19736). Usually the contents of the vacuole are discharged directly to the outside
from the pore, but occasionally there is an intervening canal, the discharge canal
(Faurk-Fremiet, 1925; Carasso, FaurC-Fremiet & Favard, 1962; Antipa, 1971).
Well-defined pores have not been seen in the contractile vacuole complexes of any
groups of the protozoa other than the ciliates. Weiss, Goodenough & Goodenough
(1977) have described intramembranous particles which attach the contractile vacuole
membrane to the plasma membrane in the flagellate Chlumydomonas. This would
ensure that the vacuolar contents are discharged at a fixed site, effectively a pore.
One consequence of the particles is that the plasma membrane lies parallel to the
underlying membrane of the contractile vacuole. A similar parallelism has been
described in some flagellates (Hibberd, 1970; Cole & Wynne, 1973; Swale, 1973;
Mignot, 19746) (PI. 36, d ) and in an amoeba (Pussard, Senaud & Pons, 1977). This
suggests that such pores may be more widely distributed. The function of these particles may be to facilitate membrane fusion and to prevent vacuolar membrane from
flooding out onto the cell surface.
(6) Types of morphological organization
Four recurring types of organization of contractile vacuole complexes are illustrated
in Text-fig. I . They are distinguished mainly by differences in the organization of the
spongiome. Type B appears to be derived from type A and both are found only in
ciliates. Types C and D are found in amoebae and flagellates. Type C appears to be
suited to a state where the contractile vacuole complex moves about within the cell.
These four main types of organization do not include all of those encountered. In a
number of flagellates and amoebae, systems of tubules, more elaborate than those
figured here, have been described (Aaronson & Behrens, 1974; Linder & Staehelin,
1977; Willaert, Stevens & Tyndall, 1978a, b) (Pls 36, c, d, s a , 6). Details of the structure of the spongiome of some groups (e.g. the polyhymenophoran ciliates) have yet
to be provided.
Trpe A. The spongiome in this type appears as an irregular network of fine tubules
(20-80 nm) (Pl. I). It has been encountered in the kinetofragminophoran ciliates (e.g.
Suctoria, Pseudomicrothorax). The contractile vacuole discharges its contents through
a permanent pore to the margins of which the vacuole is attached by microtubules.
Type B. This is found in a variety of oligohymenophoran ciliates (Schneider,
1960; Carasso et al., 1962; McKanna, 1974, 1976; Hausmann & Allen, 1977). Characteristically, the spongiome contains parallel-sided tubules with diameters of 4550 nm. Such tubules are decorated with geometrically regular arrays of 6 x 12 nm
particles on their cytoplasmic surfaces. Such decorated tubules frequently occur in
bundles. The spongiome also contains smooth-surfaced tubules with less constant
dimensions (20-80 nm). The decorated tubules are continuous with the smooth
tubules, and the latter with the contractile vacuole or with collecting canals which
may feed into the vacuole. There is a permanent pore similar to that found in
Type A. In all cases so far investigated, part of the spongiome is distensible and
appears as ampullae before the expulsion of the vacuolar contents (Patterson, 19766,
1 9 7 7 ~Patterson
;
& Sleigh, 1976).
6
D. J. PATTERSON
Type C. The spongiome forms a discrete layer around the contractile vacuole and
is made up of a large number of 20-50 nm vesicles. I n larger amoebae the vesicle layer
is surrounded by a layer of mitochondria (Mercer, 1959; Flickinger, 1973). McKanna
(1972, 1973~)has shown that the membrane of the contractile vacuole breaks up at
systole to form the vesicles which then enlarge and fuse again to form the contractile
vacuole. The membranes of the vesicles and of the vacuole both bear an irregular
coat on their cytoplasmic surfaces (PI. 4a, b).
Type D . Associated with the contractile vacuole is a layer of irregular vesicles
and tubules as noted in a variety of smaller amoebae and flagellates (Pl. 3a, 4c, d ) .
It is probable that these membranous elements enlarge and fuse to form the
contractile vacuole and that, as with Type C, the vacuolar membrane fragments at
systole.
One frequent component of contractile vacuole complexes, particularly those of
Type D, are 100 nm vesicles bearing coats of polygonally arrayed particles on their
cytoplasmic surface (Leedale, 1967; Brooker, 1971a; Hoffman, 1976; Eyden &
Vickerman, 1975; Heywood, 1978). Such coated vesicles have been described in a
variety of sites and in many cell types (Kivic & Vesk, 1974; Cole & Wynne, 1973;
Pearse, 1976; Franke, Kartenbeck & Spring, 1976; Kartenbeck, Franke & MorrC,
1977). The coat in some sites has been identified as a protein which has been called
' clathrin' (Pearse, 1976). Such vesicles are usually associated with movement and
fusion of membranous structures. The occurrence of these vesicles in certain contractile vacuole complexes may be indicative of membrane fragmentation and refusion in those organelles.
( c ) Artefacts
A detailed comparative study of contractile vacuole complexes depends on any
potentially misleading images being recognized as such. Several aspects of ultrastructural studies give cause for concern. The nature of the fixative is very important
as may be illustrated by comparing studies made on the ochromonadine flagellates
(Chrysophyceae). Contractile vacuole complexes in this group have elaborate tubular
elements (alveolate vesicles) in the spongiome when fixed in glutaraldehyde or potassium permanganate (Pl. 3 b, d ; Aaronson & Behrens, 1974; Wessel & Robinson,
1979), but these structures are not apparent when osmium tetroxide is employed as the
primary fixative (Belcher, 1969; Belcher & Swale, 1971, 1972; Hoffman, 1976;
Mignot, 1977).
In a number of studies on ciliates, regions of folded membranes are visible (Rieder,
1971; Grain, 1972; Organ, 1972; Nilsson, 1976). The tubules have diameters of over
twice that of well-preserved spongiome and for the moment ought to be regarded as the
result of the artefactual collapse of contractile vacuolar and ampullary membranes.
Indeed, the entire contractile vacuole may collapse during fixation as it frequently
appears to be flattened, fragmented or entirely absent (Elliott & Bak, 1964; Estkve,
1969; Kaneshiro & Holz, 1976; Eyden, 1977; Hausmann & Allen, 1977). The
contractile vacuole normally does collapse at systole, but the ampullae which develop
around the vacuole at this stage of the behaviour cycle of these organisms are generally
Contractile vacuoles
7
absent from electron micrographs indicating that the vacuole was not in systole when
fixed. Where such disagreement with light-microscopical observations occurs, the
results of electron microscopy are generally more suspect.
( 2 ) Behaaiour
( a) General comments and terminology
The cycle of behaviour of contractile vacuoles involves three readily identifiable
events: Diastole (filling) ; systole (vacuolar contraction) ; and expulsion (the release of
vacuolar fluid from the contractile vacuole to the outside). Expulsion occurs during
systole which is initiated by an event called rounding-up (Section VI, I ; Patterson
& Sleigh, 1976), when the vacuole changes from an irregular to a regular form. This
also marks the end of the diastolic phase of the cycle (Text-figs. 2 , 3 ) . Systole continues until diastole recommences. The duration of the cycle is called the contractile
vacuole period, and equals the sum of the diastolic period and the systolic period.
The reciprocal of this value is the contractile vacuole frequency. The rate of vacuolar
output can be obtained approximately by multiplying the frequency by the maximum
diastolic volume.
(b) Types of behaviour
A study of the patterns of behaviour of contractile vacuole complexes can provide
useful information about their organization. Eight arbitrary categories of behaviour
are listed below, six of which are illustrated in Text-fig. 2.
Type I. The contractile vacuole develops by the fusion of smaller contributory
vesicles. These are not apparent immediately after vacuolar expulsion but arise in the
mass of cytoplasm at the site occupied by the vacuole before expulsion. This mass of
cytoplasm, the systolic mass, is usually quite distinct, either because of adhering mitochondria, or because of its stiff consistency. This type of behaviour is usually associated with those contractile vacuole complexes which move around in the cell, such as
those of many amoebae (Adolph, 1926; Howland, 19276; Day, 1927; Hyman, 1936;
Ahmad & Couillard, 1974).It correlates well with morphological Type C (Text-fig. I ) .
Contributory vesicles are derived by growth and fusion of smaller vesicles produced
by fragmentation of the contractile vacuole membrane at systole (McKanna, 1972,
1973a). The term contractile vacuole is arbitrarily applied to the largest of the
contributory vesicles. During expulsion the vacuole frequently appears to be flattened
by directional cytoplasmic pressure.
Filling of the contractile vacuole from vesicles which appear after systole may also
be seen in some kinetofragminophoran ciliates in which ultrastructural studies suggest
that the vacuolar membrane does not fragment. As the vesicles do not behave independently, they can be regarded as the result of the uneven filling of the collapsed
contractile vacuole (Elliott & Bak, 1964).These structures have been called ‘apparent
vesicles ’.
Type II. The behaviour of this type is characterized by the re-formation of the
contractile vacuole from vesicles which arise in the vicinity of the contractile vacuole
as it expels its contents. There is no backflow of fluid into these vesicles (cf. Type IV).
D. J. PATTEFGON
8
1
2
3
4
5
1
2
3
4
5
Text-fig. 2. For legend see facing page.
6
Contractile vacuoles
9
This type occurs in a few flagellates and amoebae (Hyman, 1938; Patterson, 1976a),
and may be a variant of Type I with the vacuolar membrane fragmenting into visible
vesicles as it collapses.
Type III. The vacuole disappears at systole and refills by some means not apparent
with the light microscope. This type is seen in some amoebae, flagellates and kinetofragminophoran ciliates. In some species it can be regarded as a variant of Type I
in which the contributory vesicles never become large enough to be seen with the
light microscope. In others, it may be assumed that a permanent tubular spongiome
fills the collapsed contractile vacuole directly, without involving independent vesicles.
Type IF'. Ampullae appear around the contractile vacuole immediately after systole
begins (round-up) but before expulsion begins. The ampullae persist after vacuolar
collapse and after a short delay pass their contents into the vacuole as diastole begins
(Text-fig. 3 ;Patterson & Sleigh, 1976). Since the ampullae reappear in the same phase
Text-fig. 2. This figure illustrates six types of behaviour of contractile vacuole complexes
at comparable stages of their behavioural cycles: I , end of expulsion, before diastole (filling)
begins, nominally the beginning of the cycle; 2 , early diastole; 3, late diastole; 4, the beginning
of systole, as indicated by the rounding-up of the vacuole; 5 , mid systole, vacuolar contents
partly expelled; 6, end of phase of expulsion of vacuolar contents and nominally the end of the
cycle. The vacuole is shown as if viewed from above (along the axis of the pore) (T) and from
the side (S).
Type I. The contractile vacuole completely disappears during systole. Small vesicles
appear at the site of expulsion and enlarge and fuse to form the contractile vacuole.
Type 11. The contractile vacuole is replaced by a number of small vesicles during the
expulsion of its contents. These vesicles enlarge and fuse during diastole to form the new
contractile vacuole.
Type 111. The contractile vacuole simply collapses during systole, but remains intact.
During diastole the collapsed vacuole is slowly filled with fluid. Where this type of organization
occurs in ciliates, the pore and associated pellicle (dotted line) is indented during systole, i.e.
from round-up to the beginning of diastole.
Type IV. At the beginning of systole the contractile vacuole rounds up and forces some
fluid back into recurring ampullae. These ampullae persist and enlarge during systole and
may become stretched during the expulsion of vacuole contents. Diastole begins with the
ampullae discharging their contents into the vacuole with which they appear to become
confluent. The vacuole may continue to fill slowly after all the ampullae have discharged
their contents. This type of behaviour is found in many ciliates, and during the systolic part
of the cycle the pore and associated pellicle may be indented. This type of behaviour has been
noted also from some amoebae, but no indentation of the cell surface (dotted line) has been
noted.
Type V. Collecting canals may be seen to radiate away from the contractile vacuole. At the
onset of systole fluid is forced back into the most proximal portions of the canals which become
distended as ampullae. The ampullae persist and enlarge throughout systole, but release their
contents into the vacuole at the beginning of diastole. Diastole may continue with a slow
filling of the vacuole after the contents of the ampullae have been released. During systole, the
pore and associated pellicle may be indented.
Type VI. Collecting canals may be seen radiating from the contractile vacuole. At the
onset of systole a portion of the canals, separated from the vacuole by a short indistensible
region, is distended to form an ampulla. The ampullae continue to fill slowly during systole
and release their contents into the contractile vacuole at the onset of diastole. The contractile
vacuole may continue to fill slowly after the ampullae have released their contents. The pore
and adjacent pellicle may be indented during the phase of systole.
I0
D. J. PATTERSON
Text-fig. 3. Diagrammatic representation of size changes of the contractile vacuole and
associated ampullae of the contractile vacuole complexes of types IV, V and VI (Text-fig. 2)
found in ciliates. Dotted line - contractile vacuole; dashed line - ampullae; solid line - total
contents of vacuole and ampullae. The end of the cycle of behaviour is nominally determined
as being the end of the phase of expulsion (7). During the first phase of the cycle (I - the prediastolic latent period) the contractile vacuole remains empty, although the ampullae fill slowly.
Diastole begins with the phase of rapid vacuolar filling (2) during which time the ampullae
release their contents, separately, into the contractile vacuole. The vacuole may continue
to fill slowly (3) until the vacuole rounds-up (beginning of phase 4). The event of round-up
marks the end of the diastolic period and the beginning of systole. During phase 4 (the first
systolic latent period - cf. Patterson & Sleigh, r976) the vacuole remains for a short time at its
maximum diastolic volume. Phase 5 is the first systolicreduction, during which time some fluid
passes from the vacuole into the ampullae. Expulsion occurs after the second systolic latent
period (6). Only during the phase of expulsion (7) is fluid lost from the cell.
of each cycle of behaviour, they are termed ‘recurrent ’.The appearance of the ampullae
is associated with a small decrease in vacuolar volume. This is regarded as resulting
from a backflow of fluid from the contractile vacuole into the spongiome, caused by
contraction of elements around the contractile vacuole. The ampullae release their
contents only when the contractile elements relax, and not immediately after expulsion
has been completed. This type of behaviour is frequent among oligohymenophoran
and some kinetofragminophoran and polyhymenophoran ciliates (Patterson, 1976a, b).
Ultrastructural studies generally reveal that the spongiome is continuous with the
contractile vacuole at all phases and that the vacuole simply collapses during systole
(Elliott & Bak, 1964; Antipa, 1971; McKanna, 1972).
Type V . The behaviour is similar to Type IV, but the recurrent ampullae develop
in the proximal region of collecting canals. Such canals have differentiated in the
spongiome of some larger ciliates (e.g. Colpoda spp., Stentor spp., Spirostomum
SPP.).
Type VI. As with Types IV and V, some fluid is forced back from the contractile
Contractile vacuoles
I1
vacuole into recurrent ampullae during systole. The ampullae form part of distinct
collecting canals, but differ from those of Type V in being separated from the vacuole
by short, indistensible connecting canals. This type of behaviour has only been seen in
certain ciliates where the collecting canals are supported by extensions of the radial
pore microtubules. It has been described in detail for Paramecium (Patterson, 1977a)
and very closely resembles that of Tetrahymena (Patterson & Sleigh, 1976). It may
be regarded as derived from the type found in Tetrahymena (Type IV), by differentiation of the elaborate collecting canals in the spongiome.
Type VII. The contractile vacuole fills from a radiating system of irregular lacunae.
This type of organization has only been seen in some fresh-water sponges (Brauer,
1973; Weissenfels, 1974, 1975).
Type VIII. This category contains those contractile vacuole complexes with behaviour atypical of other categories. Unique behaviour patterns are exhibited by the
ciliates Paramecium putrinum ( = trichium), Loxophyllum meleagris and Haptophrya
michiganensis (King, I 928 ; MacLennan, I 944 a ;Patterson, I 976 a), by the diplomonad
flagellate Trepomonas agilis (Eyden & Vickerman, 1975)and possibly by the flagellate
Vacuolaria virescem (Heywood, 1978).
In addition to these eight types of behaviour of contractile vacuole complexes it
must be remembered that there are organisms in which no contractile vacuoles are
apparent. Freshwater organisms without contractile vacuoles, such as Pelomyxa
palustris and Loxodes spp., merit further attention as to their mechanisms of
osmoregulation.
( c ) Observation of contractile vacuoles
An accurate record of the behaviour of contractile vacuoles can best be obtained
by using cinC films (Lloyd & Beattie, 1929;Organ, 1972;Organ et al., 19686, 1969,
1972;Patterson & Sleigh, 1976;Patterson, 1977a).A quantitative estimate of vacuolar
output must take account of the frequency of expulsions and of the amount of fluid
expelled at each expulsion. This is because the amount of fluid expelled at each
expulsion is not constant, nor is there any fixed relationship between frequency of
expulsions and the rate of vacuolar output (Rifkin, 1973;Ahmad & Couillard, 1974;
Patterson, 1976a).The size of the vacuole can be measured with any of a variety of
measuring microscope oculars, but the most suitable for this purpose are imageshearing oculars (Rifkin, 1968).
The most common problem in measuring contractile vacuole activity is that the
protozoa move too fast to allow observationsto be made easily. Techniques which have
been used to slow or immobilize organisms are listed below. As the effects of these
treatments on the physiology of protozoa is poorly known, the use of sessile protozoa
is particularly attractive (Kitching, 1934 et sep.).
( I ) Deciliation, either by chloral hydrate or ethanol treatment (Grebecki & Kuznicki, 1961; Czarska, 1964; Ciccolello & Gibor, 1978). This treatment substantially
affectsthe ion and water balance of the cells (Dunlap, 1977;Ciccollelo & Gibor, 1978).
( 2 ) The use of specific immobilizing antibodies (Patterson, 1976a, Patterson &
Sleigh, 1976).This again may affect ion and water permeabilities.
I2
D. J. PATTERSON
(3) Paralysis of ciliary activity by heavy metal ions, particularly nickel (Gelei,
1935; Thomas, 1953; de Puytorac, Andrivon & Serre, 1963; Isquith & Bobrow,
1973). Such ions upset the functioning of contractile vacuole complexes (Organ, 1972;
Patterson, 1976a).
(4) Fine filamentous materials, such as lens tissue, can trap protozoa (Raze &
Schoffeniels, 1965). This is potentially very good but frequently very frustrating in
practice.
(5) Viscous materials, primarily methyl cellulose, slow the swimming of protozoa,
but methyl cellulose can affect contractile vacuoles and other structures (Raze &
Schoffeniels, 1965 ; Hjelm, 1977). Spoon, Feise & Youn (1977) have advocated the use
of the more inert polyethylene oxide.
(6) Cover-slip pressure can be used to hold moving cells for a short period of time.
This is useful for photography but pressure increases with time and disturbs the
functioning of the contractile vacuole (Patterson, 1976 a). Controlled pressure can
be exerted by micro-compressors (Spoon, 1978).
(7) Protozoa can be held in or against agar (Lee, 1941; Organ et al., 1968a; Schorr
& Boggs, 1975). Excessive pressure again disturbs the normal functioning of the contractile vacuole, particularly if the pore is occluded. Agar can also affect contractile
vacuole activity (Hogue, 1923).
(8) Marsot & Couiliard (1973) were able to immobilize ciliates by using polyanions
adsorbed onto the surface of the glass microscope slide.
(9) Rifkin & Ballentine (1976) devised a technique which they called ‘Magnetic
fettering ’ whereby they immobilized ciliates in a magnetic field, after having fed
them with a colloidal iron compound. Magnetic fields may, however, influence the
behaviour of the contractile vacuole complex (Isquith & Bobrow, 1973).
(3) Systematic survey of the occurrence and nature of contractile
vacuole complexes
Contractile vacuole complexes occur usually in protists and sponges from freshwater habitats and are less common in their marine or parasitic counterparts. Indeed,
they are absent from a variety of taxa with only parasitic (e.g. sporozoans, opalinids)
or marine (foraminifera, radiolarians) members. The particular association of
contractile vacuoles with the phagotrophic protists may be understood by considering
the obligation of such cells not to have complete, rigid, cell walls. Such cells without cell walls (cf. prokaryotes, many algae and fungi) are prone to osmotically induced
changes of volume. Contractile vacuole complexes replace cell walls in their function
of minimizing any volume changes caused by osmotic stress.
The algae exhibit a variety of states of organization, from protistan (unicellular)
to multicellular, with or without flagella, with or without entire cell walls. If multicellular algae have contractile vacuole complexes, they typically have them only
during phases in which the cells lack their rigid walls. This is true of the Chlorophyta
(Lloyd, 1928a, b) and of the Xanthophyceae (Massalski & Leedale, 1969; Moestrup,
1975). Unicellular flagellated algae, particularly in fresh-water habitats, typically
have one contractile vacuole (though sometimes more) occurring in a fixed position.
Contractile vacuoles
I3
This is true for the cryptomonads (Pl. 4d; Lucas, 1970), euglenids (PI. 3c; Hyman,
1938 ; Leedale, 1967); prasinophytes (Maiwald, 1971; Manton, 1975); Chloromonadophyceae (Schnepf & Koch, 1966; Heywood, 1978); Prymnesiophyceae (Mignot,
1 9 7 4 ~ Green
;
& Hibberd, 1977) and Chrysophyceae (Pl. 3b, d; Cole & Wynne,
1973; Aaronson & Behrens, 1974). A clear overall concept of the organization of the
spongiome in flagellates has yet to emerge, but frequently their spongiome appears
to be composed of irregular tubules and vesicles as illustrated in Text-fig. I , Type D.
One exception is an elaborate tubular spongiome found in the ochromonadine Chrysophyceae (Pl. 3b, d). Dinoflagellates are generally thought not to have contractile
vacuoles. A different kind of structure, the pusule, does occur (Dodge, 1972, 1973)
and this may be functionally analogous to the contractile vacuole complex.
Flagellates from taxa without members capable of photosynthesis (the old ' zooflagellates') often have contractile vacuoles. Exceptions to this are the parasitic metamonad, trichomonad and hypermastigid flagellates. Two diplomonad flagellate genera,
Trepomonas and Hexamita, have systems of roving vacuoles which may fulfil a
function similar to that of contractile vacuole complexes (Brugerolle, 1976; Eyden &
Vickerman, 1975). In a number of kinetoplastids the spongiome may be formed
of elaborate tubular elements (Pl. 3c) (Vickerman, 1969; Brooker, 1971a, b ; Eyden,
1977). In one species such tubules form a dense reticulum (Linder & Staehelin,
1978).
The only fungi to have contractile vacuoles are those with biflagellated zoospores
(the Oomycetes). The vacuoles are then present only in the zoospore stage (Morr6,
Mollenhauer&Blacker, 1971; Beckett, Heath & McLaughlin, 1974).The slime moulds
are more appropriately considered along with the rhizopod amoebae.
All cell types of fresh-water sponges may have contractile vacuole complexes
(Jepps, 1947). In pinacocytes the contractile vacuoles are grouped around the nucleus,
and are fed by a system of radiating lacunae (Harrison, 1972; Weissenfels, 1974,
1975; Brauer & McKanna, 1978), an arrangement not found in other taxa. The
organization of the spongiome in these organisms remains obscure.
The opalinid protozoa are not generally thought to have contractile vacuole complexes (Haye, 1930; Indira, 1960) despite certain suggestions to the contrary
(Metcalf, 1940).
The rhizopod amoebae are the only group of amoebae in which the organization of
contractile vacuole complexes has been documented. In the larger species, such as
Amoeba proteus and Chaos carolinme, the spongiome is made of a large number of
small vesicles with diameters down to 20 nm (Pl. 4a, b) (Greider, Kostir & Frajola,
1958; Mercer, 1959; Flickinger, 1973). The vesicles apparently imbibe fluid, enlarge
and fuse to form the contractile vacuole (Adolph, 1926; MacLennan, 19443; Ahmad
& Couillard, 1974). Expulsion typically occurs in the uroid, at which time the vacuolar
membrane fragments to form the small vesicles of the spongiome (McKanna, 1973a).
This type of organization is particularly suited to the contractile vacuole complex of
an organism which has no stable external layer and which is subject to very active
cytoplasmic movements. A similar morphology is found in many smaller amoebae
(Pl. 4c) (Schuster, 1963; Bhowmick, 1967; Houssay & Prenant, 1970) although a few
I4
D. J. PATTERSON
species have tubular elements in the spongiome (Vickerman, 1962; Bowers & Korn,
1968, 1969; Willaert et al., 1978a, 6). The behaviour of the complex in small species
resembles that of larger species (i.e. Type I ) (Hyman, 1936; Hopkins, 1946; Vickerman, 1962). On the whole, the contractile vacuole complexes of the amoebae of the
protostelid, dictyostelid, myxogastreid and acrasid slime moulds appear similar to
those of small rhizopod amoebae (Beckett et al., 1974; Olive, 1975).
It is among the ciliates that the contractile vacuole complex exhibits its most elaborate organization. One may speculate that this has been demanded by the relatively
large size of many ciliates, and is permitted by the presence of a stable and complex
cortex. Virtually every species so far investigated has one or more pores (as described
in Section 11. z a ) stabilized by helical pore-microtubules which wind around them,
and to which the contractile vacuole is attached by bands of radial pore-microtubules
(Pl. I U , 6 , z a ) (Schneider, 1960; Elliott & Bak, 1964; Bardele, 1968; Antipa, 1971;
Rieder, 1971; Tucker, 1971; Millecchia & Rudzinska, 1972; McKanna, 1972, 1 9 7 3 ~ ;
Kaneshiro & Holz, 1976). Spirochona gemmipara (Pl. I c ) and Paramecium putrinum
(Fahrni, 1975; Patterson, 19776) vary slightly from this basic pattern. The organization of the pore in any member of some taxa (e.g. Polyhymenophora) has yet to be
established. The spongiome is made up of a system of tubules which connect directly
to the contractile vacuole or, in many larger species, to collecting canals which radiate
from the contractile vacuole. In the kinetofragminophoran ciliates and some Oligohymenophora only irregular tubules with diameters of 20-80 nm may be seen (Pl. I a,
c, d ) (Elliott & Bak, 1964; Rieder, 1971; Millecchia & Rudzinska, 1972; Fahrni, 1975).
However, in some Oligohymenophora, particularly the peniculines and peritrichs,
decorated tubules, 45-50 nm in diameter and bearing regular arrays of 6 x 12 nm
particles on their cytoplasmic surfaces, are also apparent (Pl. zb, c, d ) (Schneider,
1960; Carasso et al., 1962; McKanna, 1972; Couch, 1973; Kaneshiro & Holz, 1976;
Hausmann & Allen, 1977). Frequently these tubules occur in tightly packed bundles
or fascicles. I n occasional species such decorated tubules contain an inner tubule
(Pl. z d ) (Kaneshiro & Holz, 1976; Patterson, 1977b). Where smooth and decorated
tubules occur, it is attractive to think that each type is involved in different functions for example the segregation of fluid from the cytoplasm and the resorption of selected
solutes from the fluid back into the cytoplasm. There is virtually no information about
the structure of contractile vacuole complexes in the Polyhymenophoran ciliates.
The behaviour of the contractile vacuole complex has now been well described for
a variety of ciliates. The contractile vacuole seems to disappear at systole, but critical
light microscopical studies and electron microscopy both suggest that the vacuole
remains intact and is simply flattened by pressure from the cytoplasm (Organ et al.,
1968b, c, 1969, 1972). Shortly before expulsion the contractile vacuole rounds up and,
in many species, a small amount of fluid is squeezed back into the spongiome (Textfig. 3). This fluid is accommodated in recurrent ampullae which had previously been
misinterpreted as independent vesicles (Taylor, 1923; King, 1928, 1933, 1935; Day,
1930; Moore, 1934). Lloyd & Beattie (1929), Patterson (19766, 1 9 7 7 ~ and
) Patterson
& Sleigh (1976) have provided detailed descriptions of this behaviour in several ciliates.
The ampullae persist until some time after the end of expulsion when they empty
Contractile vacuoles
I5
into the vacuole causing it to fill rapidly. Filling may continue slowly until the next
phase of systole begins. The ampullae are often not apparent in many kinetofragminophoran ciliates I n larger ciliates, which have collecting canals, the ampullae
develop in the proximal region of those canals.
(4) Towar& a new concept of contractile vucuoles
The most prevalent view of contractile vacuole complexes is that the contractile
vacuole, in the strict sense of the term, is the complete osmoregulatory organelle of
protozoa. This structure is often thought to be transient, in that individual contractile
vacuoles form and are completely lost at the subsequent systole. It has also been widely
suggested that contractile vacuole fluid is gained by the accretion of vesicles arising in
the cytoplasm (Taylor, 1923; King, 1928, 1933, 1935; Day, 1930; Moore, 1934;
Schnepf & Koch, 1966; Jahn, Lennartz & Fonseca, 1977; inter alia). These beliefs
have two corollaries. The first is that the fluid is packaged at cytoplasmic sites independent of the contractile vacuole and is being transported to the vacuole. Secondly,
that if the vacuole is to keep a fixed maximal size, then the continuous recruitment of
membrane due to the accretion of small vesicles with that vacuole must be offset by a
continual loss of membrane. This latter point could be explained by assuming that the
membrane of the contractile vacuole is incorporated in the plasma membrane during
the expulsion of the vacuolar contents (e.g. Hyman, 1938; Gojdics, 1953). These two
corollaries meant that the vacuole was considered as being in a state of continuing
flux and that it was directly dependent on other cytoplasmic organelles for the production of fluid.
Here an alternative concept is presented, in which the contractile vacuole complex
is considered to be a discrete organelle, functionally distinct from other cellular
organelles. It is also seen as a stable system, in that there is no massive and continuous
recruitment or loss of membrane from the complex.
This conceptual change is mainly due to the ' discovery' of a spongiome, associated
with contractile vacuoles and which may be the site of production of the fluid for the
contractile vacuole (see Section V). It thereby brings this function, together with the
more obvious ones of fluid storage and expulsion, within the activities of a single
organelle - the contractile vacuole complex. A second important factor leading to
conceptual change is the evidence from many electron-microscopic studies that
contractile vacuole membrane is not incorporated in the plasma membrane at systole.
In flagellates and ciliates the vacuole remains intact and simply collapses (Elliott & Bak,
1964;Leedale, 1967; Wessenberg & Antipa, 1968,1970; McKanna, 1972;Organ, 1972;
Heywood, 1978). The apparent involvement of vesicles in the filling process is due,
in many cases, to misinterpretation of recurrent ampullae (Patterson, 19766, Patterson,
1 9 7 7 ~Patterson
;
& Sleigh, 1976) or of uneven filling of the flattened vacuole (Elliott
& Bak, 1964). In amoebae, there is little doubt that the contractile vacuole develops by
the fusion of independent vesicles (Section 11. 36). McKanna's work (1972, 1 9 7 3 ~ )
has shown that these vesicles are not derived independently of the contractile vacuole
complex, but form by fragmentation of the membrane of the contractile vacuole
during systole.
16
D. J. PATTERSON
In some diplomonad flagellates independent vesicles appear to arise at undefined
sites in the cytoplasm and then move to the flagellar grooves where their contents are
expelled (Eyden & Vickerman, 1972). However, it could reasonably be argued that
such structures are not strictly homologous with contractile vacuole complexes.
The argument that contractile vacuole complexes are directly dependent on other
cytoplasmic organelles can be traced back to the 1920’s when it was suggested that
they were the protozoan homologues of Golgi complexes (Nassonov, 1924, I925 ;
Bowen, 1928; Gatenby, Dalton & Felix, 1955; Dalton & Felix, 1957). The two organelles have some staining properties in common (e.g. they both impregnate with
heavy metals) (King, 1935; Yamataka, 1966; McKanna, 1972). However, the suggested homology can no longer be accepted, since protozoa are now known to have
conventional Golgi complexes (Grass6 & Hollande, 1941; Esteve, 1972; Wise &
Flickinger, r976; Allen, 1978a; inter alia). I t has been suggested that the contractile
vacuole derives fluid or other materials from Golgi complex vesicles (Schnepf &
Koch, 1966; Heywood, 1978; Linder & Staehelin, 1978;inter alia). While the case
for this seems very strong in some species (e.g. Vucuoluria uirescens), in others the
argument hinges on the occurrence of IOO nm vesicles in both organelles. The widespread distribution of these vesicles in many cell types and at many cell sites (Section
11, I b) weakens this argument.
It has also been suggested that the spongiome of the contractile vacuole complex
is continuous with the endoplasmic reticulum of the cell (Pappas & Brandt, 1958;
FaurC-Fremiet, Favard & Carasso, 1962; Elliott & Bak, 1964; Mosevitch, 1965;
Organ et al., 1972; Dunham & Kropp, 1973; Harry & Finlayson, 1976). These contentions are invalidated when strict criteria are used to identify endoplasmic reticulum
(Franke, Eckert & Krein, 1971;McKanna, 1972; Hausmann & Allen, 1977).
These considerations are in conflict with suggestions that the contractile vacuole
complex is an organelle whose membrane is being continually recruited and lost and
that the contractile vacuole fluid is produced by organelles other than the contractile
vacuole complex before being passed to the complex. Rather, they suggest that
contractile vacuole complexes are stable organelles within cells, distinct from other
organelles in both morphology and function.
Only in a small number of organisms does the contractile vacuole complex not appear to be a stable structure (Trepomonus, Hexamitu), or to gain fluid from other organelles (Vumoluriu), or to be associated directly with other organelles (Spirostomum
teres) (Pl. 5 c , d ) . In such organisms the homologies of the organelles ought to be more
strictly investigated.
111. FACTORS AFFECTING ACTIVITY OF CONTRACTILE
VACUOLE COMPLEXES
Many agents can affect the activity of contractile vacuole complexes. Although we
do not yet know how the complexes work, a study of the effects of such agents may
facilitate an understanding, not only of the functioning of contractile vacuoles, but
also of several aspects of basic cell biology - such as the regulation of intracellular
activities. The effects of these agents may fall into five categories : ( I ) change in the
I7
Contractile vacuoles
rate of fluid production; (2) gain or loss of contractile vacuoles; (3) change in the
frequency of expulsions; (4) prevention of expulsion of vacuolar fluid; ( 5 ) changes in
basic behaviour patterns (Section 11, 2b).
Contractile vacuole complexes appear to be particularly sensitiveto treatments which
disturb the osmotic balance between the cell and the medium. Perturbations of
osmotic balance may be achieved by changes in the osmotic pressure of the medium,
the nature or quantity of the cytoplasmic solutes or the permeability of the cell
membrane to water. Such disturbances typically result in changes in the amount
of fluid expelled (= produced), an independent change in the frequency of contractions, or even the loss (or gain) of contractile vacuoles. A number of other treatments seem to impair the contractile machinery of the complex, thereby affecting the
frequency of vacuolar contractions and perhaps leading to arrest of expulsions while
filling continues.
The effect of osmotic agents added to the external medium have been extensively
investigated from the time of Rossbach (1872) and Hartog (1888). Such studies have
generally shown that the output of the contractile vacuole complex decreases if the
osmotic pressure of the medium is raised, and increases if the external medium
becomes more dilute (Eisenberg, 1926; Eisenberg-Hamburg, 1929; Kitching, 1934,
1936, 1938~2,1948b, 1951; Miiller, 1936; Gaw, 1 9 3 6 ~ Cosgrove
;
& Kessel, 1958;
Osanai, 1961a, b ; Stoner & Dunham, 1970; Hampton & Schwartz, 1976; inter aZia).
The extent to which the contractile vacuole complex is affected depends on both the
strength and nature of the osmolyte (Muller, 1936; Raze & Schoffeniels, 1965). The
output of the complex is not linearly related to the osmotic pressure of the medium
(Section VI. 2). The frequency of contractions is particularly sensitive to the composition of the bathing medium, but it varies independently of the total output (Stempell, 1924; Eisenberg, 1926; Eisenberg-Hamburg, 1929; Osanai, 1961a ; Czarska,
1964; McNeill & Perkins, 1972; Organ, 1972; Organ & Bovee, 1972).
In the event of a change in the osmotic balance of the cell, the output of the contractile vacuole changes rapidly. However, after a few minutes the output of the
contractile vacuole tends to return to a value similar to that preceding the shock
(Kitching, 1934, 1936, 1938a, 1948b, 1951; Seravin, 1958, 1959; Osanai, 1961a ;
Stoner, 1970; Stoner & Dunham, 1970; Pal, 1972). The basis of this return is discussed in Section V.
It may be possible to attribute the response of the contractile vacuole complex
to a variety of agents to their indirect effects on the osmotic balance of the cell.
For example, the increased output associated with higher temperatures within the
physiological range (Day, 1930; Gaw, 19363; Kitching, 1948a, b, 19546; Ahmad &
Couillard, 1974) has been suggested by Kitching to be the result of changes in
membrane permeability to water. One could equally argue that the effects of temperature on intracellular ion levels (Andrus & Giese, 1963; Klein, 1964; Hilden, 1970)
would disturb the osmotic balance of the cell. Increases in contractile vacuole activity following low doses of ultraviolet radiation (Tchakhotine, 1936) might also be
attributable to changes in membrane permeabilities. However, the arrest of vacuolar
expulsions upon exposure to higher doses of ultra-violet radiation is probably
2
B R E 55
18
D. J. PATTERSON
attributable to factors other than osmotic balance (Shirley & Finley, 1949; Brandt
& Giese, 1956; Mayer & Iverson, 1957).
Before completing the discussion of agents which might affect the osmotic balance
of the cell, mention of the drug ouabain should be made. This drug reduces the output of contractile vacuoles in ciliates (Boggs & Wade, 1972; Hampton & Schwarz,
1976), an effect which may be explained by assuming that the drug affects cytoplasmic
levels of sodium and potassium by interfering in the activity of the ion-pump, Na+,
K+-ATPase, as it does in higher cell types. This explanation is, however, not justified
since many studies (Conner, Chook & Ray, 1963; Klein & Breland, 1966; Ulsamer,
Wright, Wetzel & Korn, 1971; Andrivon, Wyroba & Patterson, 1977) have, with a
single exception (McLaughlin & Meerovitch, 1975), come to the conclusion that the
enzyme does not occur in protozoa. Certainly less direct studies have failed to show
predictable effects by ouabain on the ion levels in cells of ciliate protozoa (Andrus &
Giese, 1963; Klein, 1964; Hilden, 1969; Kropp, 1971; Prusch & Dunham, 1972).
Another category of agents change the frequency of contraction of the contractile
vacuole. Contractions are initiated by a timing mechanism whose control is independent of the process of fluid formation (Section VI). The frequency of contraction may
reflect the ionic balance in the medium (Eisenberg-Hamburg, 1929; Osanai, 1961a ;
Czarska, 1964; McNeill & Perkins, 1972; Organ, 1972). Similarities with the ionic
dependence of ciliary reversal in ciliates (Jahn, 1962) might suggest that calcium
availability could play a r61e in regulating contractile vacuole contractions. This is
supported by the inhibitory action of heavy metal ions such as nickel (Gelei, 1935;
Thomas, 1953; de Puytorac, Serre & Andrivon, 1963) and by the effects of electrical
fields (Czarska, 1964). Both treatments are known to affect calcium movements in
ciliates (Jahn, 1966; Andrivon, 1974).
Interference with vacuolar contractility may, under certain conditions, lead to
total arrest of expulsion of vacuolar fluid. Similar effects may be achieved in ciliates
if the vacuole breaks away from the pore. Under the latter circumstances, the vacuole
usually continues to fill and contractions may continue. The prevention of expulsion
has been caused by ultrasound (Mugard & Renaud, 1967), heavy water (Taylor,
Swingle, Eyring & Frost, 1933; Gaw, 1936c), cyanide (Kitching, 1936, 1938a;
Osanai, 1961c; Yamada, 1974a)and ATP (Organ et al., 1 9 6 8 ~ ) .
Many other agents are known to affect the behaviour of contractile vacuole complexes, although the reasons generally remain unclear. With the exception of a single
report relating to Amoebaproteus (Gicquaud & Couillard, 1972), there are no reports
of the microtubule-disrupting agent colchicine having any effect on contractile
vacuoles. The same is true for Cytochalasin B which affects, among other things,
actin-based contractile mechanisms; although its carrier, DMSO, influences contractile vacuoles at higher doses (Nilsson, 1974; Nilsson, Ricketts & Zeuthen, 1973).
Various agents which affect energy-generating metabolism (e.g. ATP) do influence
activity of the contractile vacuole complex. Examples of this are low oxygen levels
(Fortner, 1924; Kitching, 19393; Gittleson & Sears, r 964), a,q-dinitrophenol (Raze
& Schoffeniels, 1965; Yamada, 1974b) and cyanide (Yamada, 1974Q). 2,q-dinitrophenol is particularly interesting as it is the only drug known to interfere with the
Contractile vacuoles
I9
response of the contractile vacuole complex to osmotic shock (Stoner, 1970). The
evaluation of the effects of these, and other, agents must await a more detailed knowledge of their effects on general protozoan metabolism as well as a more thorough
knowledge of the metabolic bases for contractile vacuole activity.
Only two firm general conclusions can be drawn from these studies. The first is
that the output of the contractile vacuole complex reflects the osmotic balance of the
cell with the medium. The second is that there is a mechanism controlling the frequency of contraction and this can be influenced separately from the mechanism
of fluid production.
IV. SUGGESTED FUNCTIONS OF CONTRACTILE VACUOLE COMPLEXES
Osrnoregulation
The cytoplasm of fresh-water protists has an osmolarity in the region of 50IOO mOsm/l (Gelfan, 1928; Kitching, 1938a, 1949b, 1951;Belda, 1942; Lplvtrup &
Pigon, 1951; Schmidt-Nielsen & Schrauger, 1963; Riddick, 1968; Stoner & Dunham,
1970; Pal. 1972). Thus, fresh-water protozoa are thought to be hyperosmotic to their
medium (Table I ) , although the situation regarding marine and parasitic forms is
unclear (Kehlenbeck, Dunham & Holz, 1965; Kaneshiro, Dunham & Holz, 1969;
Kaneshiro, Holz & Dunham, 1969). Fresh-water protozoa are faced with two osmotic
problems: ( I ) the tendency towards cellular swelling as a result of the inwardly
directed osmotic gradient ; (2) the maintenance and regulation of the concentrations
of the osmotically active intracellular materials. The first problem, which arises because of the requirements of the second, may be controlled by the regulation of cellular
permeability, by the presence of internal or external cell walls which will restrict cell
volume changes, or by a mechanism which eliminates excess fluid from the cell.
Contractile vacuole complexes appear to fulfil the latter function.
Evidence that contractile vacuole complexes eliminate water from the cell to offset
the osmotic influx of water comes from several areas: ( I ) contractile vacuoles are
widespread in fresh-water organisms in which the osmotic gradient is expected to be
most severe, but are frequently absent from marine and parasitic forms (Finley, 1930;
Kitching, 1938b; Bovee, 1953; Page, 1970; inter alia); (2) contractile vacuoles occur
mainly in protists lacking rigid and entire external cell walls; and in the case of
algae and fungi with walls, contractile vacuoles only occur in the naked stages of
the life-cycle (Section 11. 3); (3) vacuolar output is depressed by an increase in the
osmotic pressure of the medium (in effect a decrease in the osmotic gradient across the
cell membrane) (Section 111); (4)the arrest of contractile vacuole activity, for example
by heavy metal ions or inhibitors of metabolic activity (Section 111), frequently results
in swelling of the cell; ( 5 ) mutants lacking contractile vacuoles may be restricted to
media with high osmotic pressures (Guillard, 1960); (6) the rate of vacuolar output
correlates well with values of the rate of water flux through cells measured by other
means (Belda, 1942; Lplvtrup & Pigon, 1951; Brauer, 1973). It is thought that the
osmoregulatory function can be achieved by a regulated production of fluid by the
contractile vacuole and the surrounding spongiome. Despite the general support for
(I)
2-2
20
D. J. PATTERSON
an osmoregulatory function (Degen, 1905; Herfs, 1922; Stempell, 1924; Kitching,
1934 et sep.) it is still hotly debated by some workers (Organ, 1972; Jahn & Boggs,
1970, 1971; Jahn, 1977).
It is now believed that the segregation of contractile vacuole fluid is achieved
by the spongiome (Section V). However, the processes of protozoan osmoregulation
are more complex than a modification of the rate of contractile vacuole fluid production. This can be illustrated by observing a cell after an osmotic shock (Textfig. 4). After initial changes, both the cell volume and the vacuolar output tend to
drift back towards their original values (Eisenberg, 1926; Seravin, 1958, 1959;
Stoner, 1970; Pal, 1972). This can only be achieved by an active change, on the part
of the protozoan cell, in the osmotic equilibrium across its cell membrane. Although
it would be possible to achieve this by modifying the permeability of the membrane
to water, it appears that there are changes in the cytoplasmic osmotic pressure due to
adjustments in the concentration of osmotically active cytoplasmic solutes (Stoner,
1970; Stoner & Dunham, 1970; Prusch, 1977). The time that it takes for this response
to lead to a new osmotic equilibrium depends on the organism, on the size of the osmotic shock, and on the specific osmolytes involved. Typical times range from 5 min
to I h. The cytoplasmic osmotic pressure may be modified by as much as 90% of
the change in external osmotic pressure.
The active response of a cell to an osmotic shock, i.e. volume regulation, is a longterm response of the cell to the osmotic pressure of the medium, and involves adjustText-fig. 4.The response of a protozoan cell to osmotic stress. When the cell is in equilibrium,
the osmotic pressure of the cytoplasm (OP,) exceeds that of the medium (OP,), the cell has a
stable volume and solute content. The osmotic influx of water into the cell is offset by the
activity of the contractile vacuole, whose total output is equal to the amount of fluid segregated into the complex minus that lost by resorption back into the cytoplasm. Values for
osmotic pressure, cell volume and fluid output are derived from Stoner (1970), Stoner &
Dunham (1970) and Dunham & Kropp (1973)~while the curves representing fluid segregation
and resorption are speculative in accordance with the model of fluid formation presented in the
text.
If the external osmotic pressure is raised so that the osmotic gradient is reversed (hyperosmotic stress), water will leave the cell which will shrink. This will cause the solutes to be
concentrated and the process will continue until isotonicity is achieved. At the same time contractile vacuole output falls, deemed to be as a consequence of a decrease in segregative activity
and of an increased resorption consequent on the raised internal osmotic pressure. After a
period of time (about 20 min) the contractile vacuole becomes active again and the cell begins
to swell. This is due to an active increase in the amount of osmotically active solutes in the
cell. This would increase the rate of resorption of fluid from the vacuole, so the increased
vacuolar output must be attributed to increased segregative activity.
In the event of a hypoosmotic shock, the osmotic gradient across the cell membrane is
increased, more fluid enters the cell and the cell swells. The activity of the contractile vacuole
increases, deemed to be due to decreased resorption consequent upon the reduced internal
osmotic pressure, and to an increased segregative activity. After aperiodof time, the cell volume
begins to fall, as does the output of the contractive vacuole. This can be most readily explained
by an active decrease in the amount of intracellular solutes which reduces the osmotic gradient
across the cell membrane. Fluid resorption from the contractile vacuole complex falls, reflecting the lower internal osmotic pressure. T h e fall in total output must consequently be attributed to a fall in the segregative activity of the complex.
Contractile vacuoles
21
<
Hyperosmotic stress
Hypoosmotic stress
...__ _ _ _ - _--__.______
__-
0 P i ..............
.......................................................................
o p e . ........
. . ....______
:. .........................................................
.............._------
.............................................
____ __ -.- - - - -.-.--.---.
I
I
'
*
Solute content
per
...............
Cell volume
...............
\
e
Contracti! ...........
vacuole output
.
. ........................................
............................
C.V. fluid ...............
segregation
C.V. flu id ................................
resorption
.................. .....................................
.............................................................
Text-fig. 4. For legend see facing page.
ment of the levels of intracellular solutes. It is a feature of a wide variety of procaryotic
and eucaryotic cell types. Specific solutes involved in re-establishing osmotic equilibrium have been called compatible solutes (Borowitzka & Brown, 1974). Typical
compatible solutes are potassium ions, free amino acids, and polyhydric alcohols
(Neelon & Bernheim, 1973; Hellebust, 1976; Brown, 1976; Hoffman, 1977). The
long-term changes in intracellular solutes after osmotic shock have been investigated
in the ciliates Tetrahymena pyriformis, Miamiensis avidus and in the amoeba Acanthamoeba castellanii (Table I ; Kaneshiro, Dunham & Holz, 1969; Larochelle &
Gagnon, 1976). In ciliates at least, the change in internal osmotic pressure is due
mainly to changes in the levels of free cytoplasmic amino acids, made available by
degradation of cellular polypeptides.
D. J. PATTERSON
22
Table I.The influence of osmotic pressure on the osmotically active cytoplasmic
constituents of two protozoa
(Values are given as amounts per fixed number of cells {thenumber equal, at the lower osmotic pressure,
to I kg cell water (Acanthamoeba) or to I kg cells ( T e t r a h y m m ) ] .Data for Tetrahymena from Stoner
(rgyo), Stoner & Dunham (1970), Dunham & Kropp (1973); and for Acanthamoeba from Pal (1972),
Drainville & Gagnon (1973) and Larochelle & Gagnun (1976, 1978).)
Tetrahymena
pyrifomis
( m ~ / 8 . 5 2x 1 0 l O cells)
&
External osmolarity
(mOsm/l.) ...
Cellular sodium
Cellular potassium
Cellular chloride
Cellular-free aminoacid pool
Internal osmolarity
(mOsm/l.)
80
6
Acanthamoeba
castellanii
(mM/4 x 1 0 1 4 cells)
&
29
9
40
I4
26
4
500
16
34
9
32
70
7
72
123
212
104
500
I80
20
56
23
In addition to the long-term changes typical of cell-volume regulation, short-term
changes have been noted in Tetrahymena pyriformis. These take the form of transient
but marked changes in sodium and potassium levels (Stoner & Dunham, 1970;
Kramherft, 1970;Kropp, 1971; Prusch, 1977). At the moment, it is not clear if these
changes are active or passive, or to what extent they contribute to the osmoregulatory
competence of the cell (see Section IV. 3).
Thus, although the contractile vacuole complex contributes to the osmoregulatory
processes of the cell, it is only one component of the osmoregulatory machinery. Its
function is to minimize volume changes resulting from the inwardly directed osmotic
gradient. The maintenance and regulation of that gradient is ensured by an independent mechanism. The presence of this overriding mechanism explains why many
authors found that protozoa did not behave as perfect osmometers (Kitching, 1934;
Hopkins, 1946;Reuter, 1963). Organ (197z),Organ 8t Bovee (1972)and Jahn (1977)
do not accept the osmotic function of the contractile vacuole complex. They argue that
the protozoan cell does not behave predictably to osmotic stress, and that the response
of the contractile vacuole complex can be correlated, not with the osmotic pressure of
the medium, but with the nature of the substance inducing the osmotic shock (Eisenberg, 1925;Czarska, 1964;Organ, 1972;Organ & Bovee, 1972;McNeili & Perkins,
1972). Their studies, however, related only to the frequency of contraction of the
vacuole, a variable which can clearly behave independently of, and therefore does not
reflect, the total output of the complex (Sections 111, VI).
( 2 ) Feeding
Increases in output of the contractile vacuole complex occur when some protozoa
feed (MacLennan, 1933; Frisch, 1937; Rudzinska & Chambers, 1951; Kitching,
1951,195zb;
Spoon, Chapman, Cheng & Zane, 1976;Canella & Rocchi-Canella, 1976).
Contractile vacuoles
23
This is presumably due to the diffusion of water from food vacuoles into the cytoplasm.
It has been calculated that water intake associated with food vacuoles accounts for
only about 5 % of the total water flux in fresh-water organisms (Dunham & Kropp,
1973; Pal, 1972). The elimination of water taken in with food vacuoles may be the
primary function of contractile vacuoles in marine protozoa (Gayevskaya, 1924;
Kitching, 1939c), although more recent evidence does suggest that marine protozoa
may also be hyperosmotic to their medium (Kaneshiro et al., 1969a, a).
( 3 ) Ion regulation
Monovalent ions
Dunham & Child (1961) suggested that cytoplasmic sodium might be maintained
at relatively low levels by its elimination via the contractile vacuole. Subsequently,
Chapman-Andresen & Dick (1962) and Kropp (1971) working with Chaos carolinense
and Tetrahymenapyriformis respectively, have shown that there is a transient increase
in total cytoplasmic sodium when the contractile vacuole activity is arrested; for
example, after a hyperosmotic shock. Prusch (1977) and Dunham & Kropp (1973)
have noted that sodium effluxes from Amoeba potem and Tetrahymenu are reduced
when the cells are subject to a hyperosmotic shock. Riddick (1968) has shown that the
sodium concentration of the contractile vacuole fluid is higher than that of the cytoplasm (Table 2). Further evidence indicates that contractile vacuoles are positively
charged with respect to the cytoplasm and this would mean that sodium must be
actively transported into them (Yamaguchi, 1960; Josefsson, 1966; Prusch, 1977). All
this favours the view that the contractile vacuole complex is involved in the sodium
economy of protozoan cells.
It is conceivable that sodium transport into the contractile vacuole complex provides
the mechanism by which fluid is segregated from the cytoplasm. However, the case
for this is weakened by two observations. First, the contractile vacuole continues
to function in deionized water (Reuter, 1963; Raze & Schoffeniels, 1965; Rifkin,
1973). Second, in high potassium media, transient changes in the cellular potassium
levels of some species follow an increase in the external osmotic pressure (with
sucrose) (Kramh~rft,1970; Patterson, 1976a; Kirst, 1977). The latter observations
indicate that arguments applied to sodium may also have to be applied to potassium.
The parts played by the contractile vacuole complex in the monovalent-ion economy
of the cells, and of the monovalent ions in the functioning of the contractile vacuole
complex need more extensive investigation.
Divalent ions
Contraction of the large heterotrich ciliate Spirostomum is dependent on local
changes of the cytoplasmic calcium concentration (Ettienne, 1970). It has been
suggestedthat the contractilevacuolehelps to regulate the levels of cytoplasmiccalcium,
perhaps by the accumulation of free calcium (Osborn, Hsung & Eisenstein, 1973;
Ettienne & Dikstein, 1974). Legrand & Prensier (1974; P1. 5c, d ) have shown that in
one species, Spirostomum teres, the contractile vacuole is continuous with the perimyonemal vesicles where calcium is believed to be stored.
D. J. PATTERSON
24
Table
Nature of contractile vacuole fluid: values for sodium (Na+) and
potassium (K+)in mM/l, and osmolarity (o.P.) as mOsm/l
2.
Contractile vacuole
I
Organism
Chaos carolinense"
Amoeba proteust
A . proteus:
Na'
19'9
-
-
,
Cytoplasm
K+
O.P.
Na+
4.6
51
32
0.6
-
68
Medium
7
-
Kf
31
-
-
O.P.
O.P.
117
< 2
I01
6
-
-
*
Riddick (1968).
Schmidt-Nielsen & Schrauger (1963).
f Mayer & Iverson (1967).
7
(4) Excretion of waste metabolites
Griffiths (1888) presented evidence that waste products of nitrogen metabolism
were excreted from protozoa via the contractile vacuole, but Weatherby (1941)
and Kitching (1967) both decided that the evidence in favour of this function was
unconvincing.
( 5 ) Respiration and circulation.
Spallanzani (1776), Flather (1919)and Ludwig (1928) proposed that the contractile
vacuole was involved in protozoan respiration. Howland (1927a) and Kitching (1956)
have justifiably argued that there was neither evidence nor need for this function to be
attributed to the contractile vacuole complex. A second early view was that the
activity of the contractile vacuole maintained cytoplasmic circulation in protozoa
(Pritchard, 1861; Taylor, 1923). This was disproved by Jennings's observation (1904)
that the contractile vacuole expelled its contents to the outside of the cell.
(6) Reproduction
In trying to equate the parts of protozoa with organs of multicellular animals,
Ehrenberg (1838) arbitrarily and unjustifiably suggested that the contractile vacuole
acted as a spermatic gland.
(7) Enzyme outlet
A variety of protozoa are known to be able to secrete digestive enzymes into the
medium (Aaronson & Patni, 1976). Elliott & Bak (1964) suggested that the contractile
vacuole might act as the route of efflux. Adequate evidence in favour of the secretion
of enzymes via the contractile vacuole has yet to be presented.
( 8 ) Excystment
Increased vacuolar activity is associated with excystment of various protozoa and,
by expelling fluid into the space around the organism, may help to force the organism
from the cyst (Carter, 1856; Holt, 1972; Jenkins, 1976).
(9) Lorica formation
The contractile vacuole is exploited in some peritrich ciliates to change the shape
of the organism and thereby help to mould the lorica (Bacon, 1973).
Contractile vacuoles
25
(10) Mucus secretion
Material derived from Golgi complexes appears to be passed to the outside of the
cell via the contractile vacuole in Vacuolaria virescens (Heywood, 1978). Th'is occurrence appears to be peculiar to this group of organisms (Chloromonadophyceae).
The contractile vacuole in this group may not be strictly homologous with those in
other organisms.
V. THE PRODUCTION OF VACUOLAR FLUID AND ITS CONTROL
Little is known of the nature of the fluid in contractile vacuoles. The results of the
small number of studies suggest that the osmotic pressure of this fluid is lower, the
concentration of sodium higher, and the concentration of potassium lower than those
of the cytoplasm (Table 2). This conclusion relies on the possibly invalid assumption
that the available measurements of the total ion content of the cytoplasm reflect
accurately the cytoplasmic-free ion concentration (Batueva & Lev, I 967;Larochelle &
Gagnon, 1978).Normally one assumes that the contractile vacuole contains an aqueous
fluid in which only inorganic substances are dissolved; this assumption, which is unsupported by experimental evidence may be misleading. Several studies indicate that
organic matter is present in the lumen of the vacuole (Brandt & Pappas, 1962;Elliott &
Bak, 1964;Antipa, 1971;
Rieder, 1971;
Lynn, 1976;Willaert etal., 19783;Heywood,
1978).
(I) The mechanism of jluid formation
In 1936 Kitching considered three means by which contractile vacuole fluid
might be segregated (water transport, the osmotic mechanism and phase separation).
T o this we must now add the mechanism of structural segregation proposed by
McKanna (1972).At the moment all mechanisms remain speculative in the absence
of sound experimental support.
( a ) Water transport
Such a mechanism involves molecular mechanisms residing within the membranes
of the contractile vacuole complex and capable of transporting individual water molecules into the complex. Lnrvtrup & Pigon (I~sI), Riddick (1968)and House (1974)
have deemed this to be a possible, but improbable mechanism.
(b) The osmotic mechanism
This mechanism implies that an osmotically active substance is accumulated in the
contractile vacuole complex so as to create an osmotic gradient into the complex.
This would then cause water to flow into the complex. Heywood (1978)suggested
that a hydrophilic organic material (the osmoticum of Vickerman & Preston (1976)?)
may be secreted into the contractile vacuole and that this material can induce a movement of water into the complex. However, a mechanism more compatible with contemporary views of how water is passed through cells and tissues, would be one in which
ions are actively transported across the membrane of the contractile vacuole complex
to produce the osmotic gradient into the complex (Oschman, Wall & Gupta, 1973).
26
D. J. PATTERSON
The ubiquitous spongiome seems a probable site for this segregation of fluid. The
fluid would be passed to the contractile vacuole either by fusion of elements of the
spongiome, or by the flow of fluid through tubules from the closed end to the contractile vacuole. It is attractive to identify sodium as the osmolyte which produces
the initial osmotic gradient as there is evidence that sodium is actively transported
into the complex (Section IV. 3). However, such a suggestion would be premature, as
this mechanism is inconsistent with the observation that the contractile vacuole complex remains fully functional when cells are placed in distilled water (Reuter, 1963;
Raze & Schoffeniels, 1965; Rifkin, 1973).
As stated so far, such a mechanism could only create a fluid hyperosmotic to, or
isosmotic with, the cytoplasm. As this is not the case (Table 2) it is necessary to
assume that resorption of the osmolyte must occur. If this is to be effective, it must be
at a time or place where the membrane is relatively impermeable to water. It is
attractive to think of the decorated and smooth areas of the spongiome of oligohymenophoran ciliate contractile vacuole complexes (Type B) as being the segregative
and resorptive areas respectively, although there is no direct evidence for this. SchmidtNielsen & Schrauger (1963) have proposed that, in the contractile vacuole complex
of an amoeba, the segregation occurred in the vesicular spongiome and that resorption
occurred from the vacuole itself. Riddick (1968), however, was unable to confirm
that there was any reduction in the osmotic pressure of the fluid within the contractile
vacuole.
In most organisms the contractile vacuole would seem to act simply as a store
for fluid which has been elaborated elsewhere in the complex. Since vacuolar fluid is
hypo osmotic to the cytoplasm, the membrane of the contractile vacuole would be
expected to have a very low permeability to water.
Phase separation
Proponents of this mechanism tend to take the unconventional, but not necessarily
unjustified, stance of treating cell cytoplasm as a colloid and not as a fluid (Organ,
1972; Jahn & Boggs, 1970, 1971; Jahn, 1977). It is proposed that fluid, such as the
contractile vacuole fluid, could be segregated by some kind of conformational rearrangement of this colloid. It is suggested that the hyaline cap of amoebae might
be produced by such a mechanism. Differences between the concentrations of ions
in cytoplasm and vacuolar fluid present no difficulties to this hypothesis as it has
already been established that the concentration of an individual ion species in one cell
can differ between regions in which the cytoplasm is in different conformational
states (Anderson, 1964).
The case for this mechanism has not yet been well formulated and no r61e for the
membranous spongiome has been articulated. T o some extent these proposals were
generated in order to explain anomalous results regarding the effect of different ions
on the frequency of vacuolar contractions (Czarska, 1964; Organ, 1972; McNeill &
Perkins, 1972). The need for such an explanation was derived from the mistaken
assumption that contractile vacuole frequency could be taken as an estimate of water
flux through the protozoan cell and thus of the rate of fluid production.
(c)
Contractile vacuoles
27
( d ) Structural segregation
McKanna (1972, 1974, 1976) has suggested that the cytoplasmic side of the membrane of the spongiome of all contractile vacuole complexes is structured in such a
way as to impose an order upon the adjacent cytoplasmic water, thereby favouring a
flow of water into the spongiome. This proposition draws upon an unconventional
view of the physical nature of the cytoplasm, and exploits a novel concept of water
flux which cannot be considered in detail without corroborative evidence. McKanna’s
view that the particles decorating the cytoplasmic surfaces of membranes of the spongiome of all contractile vacuole complexes are morphologically and functionally
homologous appears to be rather generous. Well-ordered arrays of particles have only
been described in the oligohymenophoran ciliates and in two flagellates (Pl. zb, c, d,
5b, Section 11, I) (Wessel & Robinson, 1979) but not yet elsewhere. Elaborations on
the surface of the vesicles of the spongiome in amoebae (Pl. 4b) might best be regarded
as artefacts (Section 11, Ia). A more critical assessment of such elaborations of the
membranes must be undertaken before McKanna’s proposals can be substantiated.
It should also be noted that, even if it is ‘proved’ that to view the cytoplasm as a
colloid is more reasonable than to view it as a fluid, mechanisms involving membraneassociated pumps are not invalidated thereby.
(2) Control
of jluid production
If we accept that contractile vacuole complexes expel fluid from cells to prevent
the swelling which would otherwise result from the influx of water driven by an
inwardly directed osmotic gradient, then it is apparent that the output must be
regulated to respond to changes in transcellular water flux. Such regulation must
accommodate sudden changes in flux (for example after an osmotic shock) as well as
gradual changes (as a component of the cellular homeostatic mechanisms).
Text-fig. 4 was constructed in an attempt to establish any correlations between
contractile vacuole output and other variables, and thereby investigate how vacuolar
output might be regulated. It describes the response of the cell to osmotic shocks.
Much of the information is derived from studies on Tetrahymena pyriformis (Stoner,
1970; Stoner & Dunham, 1970; Dunham & Kropp, 1973). This diagram is also
partly speculative in that the production of fluid is thought of as occurring in three
steps. These are: (I) the segregation of fluid across a semipermeable membrane into
the spongiome by means of an osmotic gradient established by membrane-associated
active pumps transporting one or more solutes; (2) the fluid formed will be hyperosmotic to, or isosmotic with, the cytoplasm and is converted to a hypo-osmotic fluid
by resorption of solutes; and (3) storage of the hypo-osmotic fluid in the contractile
vacuole until expulsion. These functions may occur at different sites in the complex,
or at the same site, the function of which changes with time. Fluid will be lost from the
contractile vacuole complex to the cytoplasm by osmosis (resorption) during the second
and third phases. It is assumed that the rate of fluid resorption will be proportional
to the osmotic pressure of the cytoplasm.
Text-fig. 4 can be used to consider the sequence of events following an osmotic
shock. In the case of a hyperosmotic shock, the external osmotic pressure suddenly
28
D. J. PATTERSON
rises to exceed that of the cytoplasm. The resulting osmotic gradient draws water out
of the cell which shrinks and thereby concentrates its osmotically active cytoplasmic
constituents. This process will continue until isotonicity is achieved. During this
time contractile vacuole activity stops. This is deemed to be due in part to the increased resorption of fluid from the complex consequent upon the rise in cytoplasmic
osmotic pressure, and also to a decrease in the rate of fluid segregation. As its major
response to osmotic stress, the cell begins to increase the levels of the compatible
solutes in the cytoplasm (see Section IV, I). This raises the cytoplasmic osmotic
pressure until it exceeds that of the external medium and water starts to flow back
into the cell. The cell swells and the contractile vacuole becomes active again. If the
rate of fluid resorption from the complex is related to the cytoplasmic osmotic pressure,
then fluid resorption must increase during the period. Thus, the overall increase
in fluid production must involve an increase in the rate of fluid segregation.
The response of protozoan cells to a fall in the external osmotic pressure is less well
documented. There is a transient swelling of the cell and increase in contractile
vacuole output. Both tend to return to values near their initial status. It is presumed
that this osmotic adaptation involves a decrease in cytoplasmic solutes and an overall
decrease in the segregative activity of the contractile vacuole complex, i.e. the converse of the response to a hyperosmotic shock.
According to these suggestions,the changes in vacuolar output reflect active changes
in the rate of fluid segregation. This proposition has yet to be tested.
It is clear that the protozoan cell is capable of maintaining a fairly constant cell
volume, and also that it is able to regulate the efflux of fluid via the contractile vacuole
complex. How does the cell obtain the information about the rate of flow of water
into the cell? It is possible to envisage a variety of variables, one or more of which
could provide information about the osmotic status of the cell. These would include:
osmotic gradients between cytoplasm and organelles ; cell hydrostatic pressure, membrane tension; concentration of specific cytoplasmic solutes ; and the osmotic gradient
across the plasma membrane.
It is difficult to imagine how the cell could monitor the osmotic gradient across
the cell membrane directly. It is also obvious that the activity of the contractile
vacuole complex does not directly reflect the osmotic pressure of the medium - as
noted above, the protozoan cell does not behave as a perfect osmometer. Kitching
(1951)favoured a control mechanism associated with changes in cell volume. He
estimated that a suctorian could adjust the output of the contractile vacuole in response
to volume changes of as little as 1.5%. Changes in cell volume would probably be
accompanied by changes in cell hydrostatic pressure, membrane tension and in the
concentration of specific cytoplasmic solutes.
All three factors are attractive as possible control mechanisms. This is particularly
true of specific solutes such as the metallic cations, as these show transient changes
in their concentration following osmotic shock (Kramhoft, 1970; Stoner, 1970;
Stoner & Dunham, 1970;Dunham & Kropp, 1973). Furthermore, the activity of the
contractile vacuole complex is particularly sensitive to external sodium concentrations (Raze & Schoffeniels, 1965;Rifkin, 1969;Hampton & Schwartz, 1976). How-
Contractile vacuoles
29
ever, the use of such ions for control would seem improbable as the normal processes
of ion regulation would need to be subverted during osmotic stress; and also because
the cellular levels of these ions are affected by other factors not known to disturb
contractile vacuole activity (Dunham & Child, 1961; Andrus & Giese, 1963; Holm,
1970).
It is not yet possible to be more precise about how contractile vacuole output may
be controlled. This could be an exciting area for investigation particularly as the
3 Reznikoff (I926), Howland &
microinjection techniques employed by Chambers i
Pollack (1927b), and Osanai (1961d, 1963) would provide a means of distinguishing
between a control mechanism based on specific solutes and one based on hydrostatic
pressure or membrane tension.
Even if one does detect the agents which modulate the activity of the contractile
vacuole complex, the method by which it responds has still to be elucidated. The
number of segregative sites may increase, or the rate of segregation may change, as
may the rate of resorption by adjustment of membrane permeabilities. McKanna
(1972) has already presented some evidence that the response involves hypertrophy
of the spongiome.
VI. VACUOLAR CONTRACTILITY
( I ) The case for contractility
The contractile nature of the contractile vacuole was not seriously questioned until
the studies of Jahn et al. (1964) and Wigg et al. (1967). These authors noted that,
as the contents of the contractile vacuole of Amoeba proteus were being expelled, the
contractile vacuole collapsed. They argued that the vacuole should remain spherical
if the expulsion of vacuolar contents was achieved by vacuolar contractility. They
felt the collapse indicated that discharge was passive and so recommended the term
‘water expulsion vesicle’ to replace the apparently misleading ‘contractile vacuole’.
Although the ‘collapse’ of the vacuole shows that vacuolar fluid is expelled by
cytoplasmic pressure, this does not preclude the possibility that the vacuole, or associated structures, are contractile. Under conditions where there is resistance against
the expulsion of fluid, i.e. in species with thin or tortuous contractile vacuole pores
(Parameciumputrinum and Suctoria) or where the contractile vacuole pore is occluded,
the contractile vacuole does remain spherical throughout systole (Patterson, 1976a,
1977 b). Under these conditions, hydrostatic pressure of the cytoplasm appears inadequate to effect expulsion and vacuolar contractility is apparently involved. A number of phenomena confirm the view that contractile vacuoles or elements intimately
associated with them are contractile. One such is the tendency of contractile vacuoles
;
& Burton,
to take on a rounded shape at the onset of systole (Kitching, 1 9 5 4 ~Cameron
1960; Zaman, 1970; Patterson & Sleigh, 1976; Patterson, 1 9 7 7 ~ )Such
.
a rounded
shape can only be due to a force being applied evenly to the inner or outer surface
of the membrane of the contractile vacuole. It it is assumed that the membrane of
the contractile vacuole is indistensible, then the rounded shape might be due to filling
the vacuole until it becomes turgid. However, if this were the case, the vacuole should
become rounded at the same size in every cycle of behaviour. This is not the case
D. J. PATTERSON
30
and if expulsion fails, the contractile vacuole will relax, enlarge and become rounded
at a larger size (Dunham & Stoner, 1969; Patterson, 1976a; Patterson & Sleigh,
1976). Consequently, I have concluded that the rounding-up is due to the action of
contractile elements that ensheath the vacuole (Patterson & Sleigh, 1976; Patterson,
1976b, 1977U). Prusch & Dunham (1970) were able to induce contraction of contractile vacuoles isolated from Amoeba proteus thus supporting the view that contraction
is an active property of the organelle.
In relation to Text-fig. 3, contraction is thought to commence at the beginning of
phase 4 when it causes the vacuole to round-up and presents sufficient resistance to
prevent further filling. Continued contraction of the elements around the vacuole may
cause fluid to be forced back into the spongiome which may then dilate and appear
as ampullae (phase 5). Later, the continued contraction occludes the connections with
the spongiome and thereby prevents further backflow (phase 6 ) . Finally, by a means
unknown, the diaphragm is perforated (Organ et al., 19683) and expulsion occurs
(phase 7) ;achieved either by cytoplasmic hydrostatic pressure or by vacuolar contractility or bya combination of both. The delayin the release of the contents of the ampullae until some time after the end of expulsion may be attributed to the continued
contraction of the contractile elements and the continued occlusion of the connections
of the spongiome with the vacuole.
We have, as yet, little information regarding the nature of the contractile structures.
Although actin and actin-myosin systems are extensively involved in cell contractility,
there is neither biochemical nor ultrastructural evidence to suggest any association
of these molecules with contractile vacuoles. Suggestions have been made that the
generation of the contractile force is associated with microtubules (Schneider, 1960;
Elliott & Bak, 1964; Czarska, 1964; McKanna, 1972; Hausmann & Allen, 1977).
Only the contractile vacuole complexes in ciliates incorporate microtubular structures,
and there the distribution of microtubules, as bands on the poreward hemisphere of
of the vacuole, is unlikely to account for the symmetrical rounding-up of the vacuole.
Control of the contractile process
Evidence was presented in Section I11 to show that the frequency of contractions
can be modified independently of the rate of fluid production. It is relatively easy to
prevent the expulsion of the vacuolar contents in ciliates, for example by allowing a
cover-slip to press against the pore and under these circumstances periodic contractions of the vacuole are apparent. This contradicts the stated, or implied, view that
contraction is initiated when the contractile vacuole reaches a critical size (Pappas
& Brandt, 1958; Organ et al., 1969, 1972). Evidence like this supports the views of
Kitching (1956) and of Dunham & Stoner (1969) that contractions are controlled by
an independent timing mechanism. The mechanism is probably associated with
each vacuole, as different contractile vacuoles within one organism may contract
with different frequencies (Unger, 1926; Frisch, 1937).
Very little information is available to suggest how the frequency of contractions
is regulated. It has been noted (Section 111) that the frequency is sensitive to the
ratio of cations in the medium and it has been directly correlated with the Gibbs(2)
Contractile vacuoles
31
Donnan ratio (the ratio of the concentration of monovalent cations to the square root
of the concentration of divalent cations) (Organ, 1972; McNeill & Perkins, 1972).
The evidence of this last point is still unconvincing, but if correct, it would implicate
calcium as the controlling agent since the intracellular availability of this ion is directly
related to the Gibbs-Donnan ratio of ions in the medium (Jahn, 1962).
VII. ONTOGENY AND PHYLOGENY OF CONTRACTILE VACUOLE COMPLEXES
We know very little about either the individual or the evolutionary genesis and
development of contractile vacuole complexes. Indeed, we do not even know to what
extent we are justified in treating all contractile vacuoles as homologous structures,
i.e. as having evolved from a common ancestral contractile vacuole.
It would appear that contractile vacuoles evolved within the eukaryotes. Prokaryotes
lack these organelles and they are also absent from certain groups of unicellular organisms that exhibit primitive features, such as the red algae and the dinoflagellates.
It is not possible to say whether the widespread occurrence of contractile vacuole
complexes among most other groups of free-living unicellular protists should be taken
as evidence of their appearance early in the evolutionary history of eukaryotes, or as
the result of convergent evolution. The pressure that might have led to such convergent evolution could have been the need to have some means of limiting osmotically
induced volume changes in evolutionary lineages where rigid cell walls had been
relinquished to permit phagotrophy. It seems probable that contractile vacuole complexes evolved as an elaboration of some pre-existing membranous system, such as
food vacuoles or the Golgi complex.
Very little information is available about the replication of contractile vacuole
complexes when cells divide. Gelei (1938) and Rieder (1971) believe that the spongiome of new complexes in Didinium is derived from the spongiome of the pre-existing
contractile vacuole complex. In some ciliates, the contractile vacuole pores of cells
entering division may be lost, and all succeeding pores develop de nmo, while in others
the pre-existing pores persist (Wichterman, 1953; Diller, 1974). The pore forms part
of the cortex in ciliates initially appearing alongside a cortical kinetosome (Ng, 1979).
Once formed it can move relative to other structures (King, 1954; Tucker, 1971;
Suhama, 1973; Kaneda & Hanson, 1974). The ultimate position of the contractile
vacuole pore is governed by factors similar to those regulating the final position of
other cortical components. An approximate position is assured by proportional distancing along morphogenetic gradients (Nanney, 1972; Lynn & Tucker, 1976) and
a more precise position is achieved by structural guidance, i.e. being located relative
to pre-existing cortical structures (Ng, 1977; Ng & Frankel, 1977).
In some amoebae the contractile vacuole complexes seem to disappear when cells
are dividing (Botsford, 1926; de Saedeleer & Wolff, 1931b ; Pussard, 1964). However,
it is not possible to be sure that the complexes subsequently develop de novo in these
organisms, as submicroscopic components of the spongiome may persist throughout
the processes of cellular division.
In Spirogyra contractile vacuole complexes differentiate during conjugation and
persist for only a short part of the life cycle (Lloyd, 1928b).
32
D. J. PATTERSON
Although our knowledge of contractile vacuole genesis and replication remains
patchy, it would seem to be an attractive and amenable area for research into organellar
differentiation and regulation.
VIII. SUMMARY
(I) Contractile vacuoles are subcellular structures which are recognized by their
behaviour of slowly filling with fluid and periodically expelling this fluid from the
cell. Typically, they occur in unicellular eukaryotic organisms which live in media of
low or varying osmotic pressure and which lack a complete rigid cell wall.
(2) The vacuoles themselves are only part of a more extensive organelle, the contractile vacuole complex. This includes a contractile vacuole, a membranous spongiome and, in some cases a distinct pore. This organelle appears to be functionally
discrete within the cell, and is a stable and usually permanent structure. Its detailed
organization, as determined by electron microscopy and study of its behaviour shows
significant differences in different taxa of protists and various types of organization are
described.
(3) The behaviour of the contractile vacuole is sensitive to changes in the physical
conditions and composition of the external medium. The effect of such changes may
be to prevent expulsion, change the frequency and/or the output of the vacuole or
change the pattern of behaviour of the complex. The organelle is particularly sensitive to those factors that change the osmotic gradient across the cell membrane or
modify the permeability of the cell to water.
(4)The primary function of the contractile vacuole complex appears to be to
expel fluid from the cell, thereby preventing cell swelling which would otherwise
result from the inwardly directed osmotic gradient. This gradient is maintained by a
mechanism independent of the contractile vacuole complex, the cell volume regulatory
mechanism,which also serves to re-establish the osmoticgradient after an osmoticshock.
(5) Contractile vacuoles are capable of active contraction. Although the mechanism
and its control are not known, it is clear that contractions are controlled by a modifiable timing mechanism which is independent of the process of fluid production.
(6) The way in which fluid is produced is not known but a three-step model is
presented. According to this model, fluid is segregated from the cytoplasm by means
of local osmotic gradients across the membranes of the spongiome which surrounds
the contractile vacuole. Solutes are resorbed to produce a fluid hypo-osmotic to the
cytoplasm. This fluid is stored in the contractile vacuole until expulsion.
IX. ACKNOWLEDGEMENTS
I would like to thank the following authors and publishers who have so graciously made available
micrographs published here as Plates 1-5. S. Aaronson (Pl. 3 b, published originally as Figs 5 and 6
in Aaronson, S. & Behrens, U. J., J. Cell. Sci. 14 (1974): 1-9. Reprinted with permission of the
publishers); B. E. Brooker (Pl. 3 c , published originally as Fig. 39 in Brooker, B. E., 2. Zellforsch. 116
(1971):532-563. Reprinted with permission of the publishers); J. Fahrni (PI. IC); K. Hausmann
(Pls I d, 2 a, b, 4d); D . J. Hibberd Cpl. 3 d); G.F.Leedale (PI. 3 a); B. Legrand (P15 c, d);J. C. Linder
(P1 ga, b); J. A. McKanna (Pl. 4a); M. J. Ord (Pl. 46); F. Page (PI. 4c); M. A. Rudzinska (P1 la, b ) .
I wish also to express my gratitude to A. E. Dorey and M. A. Sleigh for their helpful criticisms of
the manuscript.
33
Contractile vacuoles
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EXPLANATION OF PLATES
PLATEI
( a ) Electron micrograph showing the contractile vacuole complex in the suctorian ciliate Tokophrya
infusionum. The vacuole opens to the outside by means of a permanent pore from which radial bands of
microtubules extend to the poreward surface of the contractile vacuole. The spongiome is indistinct,
but appears to be comprised of irregular tubules ( x 19000). (b) Electron micrograph showing the contractile vacuole pore of Tokophrya infusionum in cross section. Microtubules associated with the membrane of the pore as well as microtubules extending from the pore are visible. Note the close association
with a group of (barren) kinetosomes ( x42000). (c) Electron micrograph of the contractile vacuole
region of Spirochona gemmipara, a chonotrich ciliate. There does not appear to be a contractile vacuole
at the base of the pore although there are irregular tubular elements of spongiome. The pore is supported
by longitudinal microtubules and microtubules also radiate from the base of the pore into the cytoplasm
( x 37000). ( d )An electron micrograph showing a portion of the contractile vacuole complex of Pseudomicrothorax dubius. The contractile vacuole, to the right, is surrounded by a spongiome of irregular
tubules. Elements of rough endoplasmic reticulum are also present ( x 19300).
PLATE
2
( a ) Electron micrograph of an oblique section through the contractile vacuole pore of Paramecium
caudatum showing the presence of microtubules winding around the pore as well as bands of microtubules extending away from the pore ( x 30000). (b) Electron micrograph of a cross section of a collecting canal of the spongiome of Paramecium caudatum. The canal, somewhat collapsed, is supported by
small groups of microtubules. Smooth elements of the spongiome surround the collecting canal and
connect to it. Beyond the smooth tubules is the orthotubular system, regular arrays of decorated
tubules ( x 55 coo). (c) Electron micrograph of part of the contractile vacuole complex in Paramecium
putrinum. Smooth elements of the spongiome connect directly with the contractile vacuole. Decorated
tubules arranged in fascicles (the orthotubular system) lie beyond the smooth tubules, and appear to
have connections with the smooth tubules ( x 52000). (d)Part of the orthotubular system of Paramecium
putrinum showing the fascicles of decorated tubules. Some decorated tubules contain an inner tubule
( X 90500).
PLATE3
( a ) Electron micrograph of the Golgi complex and contractile vacuole region (lower left) in Euglena
spirogyra. The contractile vacuole is collapsed and its position is marked by the large number of irregular tubular and vesicular elements. Regular coated vesicles are also common (arrows) ( x 19 500).
(b) Electron micrograph of the contractile vacuole complex in Poterioochromonas malhametlsis. The
spongiome is made up of flattened tubular sacs which bear coatings on both sides of the membrane
( x 47400; inset, x 81200). (c) Electron micrograph of the contractile vacuole complex in the trypanosomatid flagellate Crithidiafascinrlata. The spongiome is made up of regular tubular elements ( x 90000).
( d ) The contractile vacuole complex of Ochromonus tuberculata. The tubular spongiome resembles that
of P. malhamensis (above). T h e contractile vacuole membrane and plasma membrane parallel each
other for a short distance, and in this region the plasma membrane bears a highIy differentiated surface
coat ( x I 5 000).
46
D. J. PATTERSON
PLATE4
(a)Electron micrograph of the contractile vacuole complex in Amoeba proteus. The contractile vacuole
(centre) is surrounded by a layer of small vesicles and then by a layer of mitochondria. (b) Electron
micrograph of the contractile vacuole in Amoeba proteus showing part of the contractile vacuole and
associated vesicles of the spongiome. T h e vesicles have irregular coats on their cytoplasmic surfaces
( x 50000). (c) The contractile vacuole complex in VexeZ1;fera bacillipedes, a small amoeba. The spongiome contains irregular vesicles and tubules ( x 30000). (d) The contractile vacuole complex in an
unidentified cryptomonad flagellate. T h e spongiome contains irregular tubular and vesicular elements,
as well as some regular coated vesicles (arrows) ( x 31 000).
PLATE
5
(a)Electron micrograph of spongiome of contractile vacuole complex of the trypanosomatid flagellate,
Leptomonas sp. The spongiome is made up of an elaborate reticulum of tubules. Coated elements may
be seen at the upper periphery of the spongiome ( x 45000). (b) Isolated element of spongiome from
Leptommas sp. showing the presence of regularly arrayed particles associated with the membrane
( x 126000).(c, d ) Two electron micrographs from a sequence of continuous sections of the ccntractile
vacuole region of the ciliate Spirostomum teres. The contractile vacuole is to the left. In (c), the contractile myonemes (arrowed) may be seen to be surrounded by perimyonemal vesicles, but in (d) the vesicles
may be seen to be continuous with the contractile vacuole ( x 2s 000).