J. Cell Sci. 5, 65-91 (1969)
65
Printed in Great Britain
THE CLASSES OF ENDOSYMBIONT OF
PARAMECIUM AURELIA
G. H. BEALE AND A. JURAND
Institute of Animal Genetics, Edinburgh 9, Scotland
AND J. R. PREER
Department of Zoology, Indiana University, Bloomington, Indiana 47401, U.S.A.
SUMMARY
The endosymbionts of Paramecium aurelia appear to consist of a number of different Gramnegative bacteria which have come to live within many strains of paramecia. It is not known
whether in nature this relationship is mutually beneficial or not. The symbionts from one
paramecium may kill other paramecia lacking that kind of symbiont. We identify the following
classes of endosymbiotic organisms. First, kappa particles (found in P. aurelia, syngens 2 and 4)
ordinarily contain highly characteristic refractile, or R, bodies, which are associated with the
production of a toxin which kills sensitive paramecia. In certain mutants of kappa found in the
laboratory both R bodies and ability to kill have been lost. Second, mu particles (in syngens i, 2
and 8) produce the phenomenon of mate-killing. Third, lambda (syngens 4 and 8) and sigma
particles (syngen 2) are very large, flagellated organisms which kill only paramecia of syngens
3, 5 and 9, and are enclosed in membrane-bound vacuoles. Fourth, gamma particles (syngen 8)
are minute endosymbionts, surrounded by an additional membrane resembling endoplasmic
reticulum. They have strong killing activity but no R bodies. Fifth, delta particles (syngens 1
and 6) possess a dense layer covering the outer membrane. At least one of the two known stocks
is a killer. Sixth, nu particles are a heterogeneous group of particles (syngens 2 and 5) which do
not kill or possess distinctive morphological characteristics. Seventh, alpha particles (syngen 2)
are the only known nuclear symbionts of P. aurelia; they are found in the macronucleus.
Alpha is also exceptional in being the only particle which is highly infectious, though certain of
the other symbionts can also be taken up by paramecia lacking them, under special conditions.
INTRODUCTION
The object of this paper is to survey the different kinds of endosymbiont which
grow in the cytoplasm or macronucleus of Paramecium aurelia. In using the word
'endosymbiont', we simply mean one organism living within the cells of another, and
do not imply anything about the interactions between host and symbiont, whether
beneficial or harmful. Knowledge of the symbionts of P. aurelia stems from the discovery by Sonneborn (1938a), that certain strains of the ciliate are 'killers'. The
killer paramecia were originally inferred to contain kappa particles in the cytoplasm
from the inheritance of the killer trait (Sonneborn, 1945). It was shown that, due to
the presence of kappa, a toxic material, at first called 'paramecin', was released into
the water, and sensitive paramecia in the vicinity were damaged or killed.
Later, kappa particles were identified under the light microscope by examination of
fixed and stained paramecia (Preer, 1950) and by observations with the phase-contrast
microscope of unstained crushed paramecia (Preer & Stark, 1953).
5
Cell Sci. 5
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G. H. Beak, A. Jurand and J. R. Freer
Killer paramecia and kappa particles were at first considered to be of interest as
illustrations of non-Mendelian heredity. However, in the course of time attention has
been concentrated more on the nature of the particles themselves. By 1959 such
details of the structure and composition of kappa (and related) particles as were then
known, showed that they were as large and complex as bacteria, though kappa particles
had some peculiar features not previously known in typical bacteria (Sonneborn, 1959).
After the initial discovery of the first killer paramecia, other types were found.
Siegel (1953) described the 'mate-killers', and Schneller (1958) described the 'rapidlysis' killers. These different types of killer paramecia were each found to contain
distinct cytoplasmic particles, those in mate-killers being called 'mu', and those in
rapid-lysis killers 'lambda'. Moreover, a number of variants (or 'mutants') of the
original kappa particles were found (Dippell, 1950). One variant discovered by
Hanson (1954) was denoted 'pi'. These pi particles were distinguished from the
original kappas by the loss of killing properties associated with the particles.
By 1956 it had become clear that kappa and similar particles were by no means
uncommon constitutents of paramecia. Sonneborn (1956) estimated that at least 30%
of stocks of certain syngens of P. aurelia when first collected from nature were killers
or mate-killers.
It has been known for many years that if paramecia are washed in bacteria-free
medium, crushed and observed in the phase-contrast microscope the presence of
endosymbionts may be ascertained, irrespective of whether there is any killing effect
or not (Preer & Stark, 1953). More recently a simple and much more rapid technique
has been devised whereby one can quickly see whether kappa or other particles are
present in a paramecium (Beale & Jurand, 1966). It is now known that a substantial
proportion of wild strains of paramecia contain symbionts in the cytoplasm, and some
even in the macronucleus. So many types have now come to light that we feel it is
desirable to attempt a comparative survey and an assessment of the homologies and
significance of the symbionts.
It should be stressed that this paper is in no sense a complete account of our knowledge of any of the particles. Fuller information about some of them (kappa, mu,
lambda) is given in an earlier review by Sonneborn (1959).
MATERIALS AND METHODS
Techniques used in these studies will be found in the following papers: (1) details
of the methods used in collecting and cultivating paramecia are given by Sonneborn
(1950); (2) the demonstration of killing (including mate-killing phenomena) is
described by Sonneborn (1959); (3) for recognition of endosymbionts by light microscopy of stained whole paramecia see Beale & Jurand (1966) and by phase-contrast
microscopy of crushed paramecia see Preer & Stark (1953); (4) for techniques for
electron microscopy see Jurand & Preer (1968).
The material used in the various studies referred to in this paper belongs to a number
of the syngens of P. aurelia. (The term syngen was introduced by Sonneborn (1957)
to refer to a group of stocks between which conjugation may occur and result in
Endosymbionts of Paramecium
67
viable progeny.) Table 1 contains a list of the stocks referred to in this paper, a stock
being the progeny of a single individual collected from nature. Although in this
account we refer to symbiont-bearing stocks in the syngens in which they are known
(syngens 1, 2, 4, 5, 6 and 8), not all the known symbiont-bearing stocks in these syngens
appear in the table, and some other symbionts occur in syngens not yet described.
Stocks which bear numbers in the series 1-350 were kindly supplied by Dr T. M.
Sonneborn; those with numbers above 500 are from our own collections.
Table 1. Syngens, stocks and symbionts referred to in this paper
Syngen
Stock no.
Place collected
54°
548
Mexico
Los Angeles, California, U.S.A.
San Francisco, California, U.S.A.
Monterey, California, U.S.A.
Pisa, Italy
N. Carolina, U.S.A.
Milan, Italy
Georgia, U.S.S.R.
Bloomington, Indiana, U.S.A.
55i
555
561
7
2
562
57O
114
IOIO
Hu 35-1
4
5
51
239
87
314
6
8
225
216
229
299
214
565
Type of symbiont
Mu
Mu
Mu
Mu
Delta
Kappa
Kappa and alpha
Mu
Sigma
Cove Lake, Tennessee, U.S.A.
Nu
Edinburgh, Scotland
Spencer, Indiana, U.S.A.
Holmes Co., Florida, U.S.A.
Philadelphia, U.S.A.
Pike Co., Illinois, U.S.A.
Florida, U.S.A.
Florida, U.S.A.
Florida, U.S.A.
Panama
Florida, U.S.A.
Uganda
Nu
Kappa
Lambda
Nu
Nu
Delta
Lambda
Lambda
Lambda
Gamma
Gamma
THE CLASSES OF SYMBIONTS
Introduction
In this survey we shall continue to use the system of denoting the principal types of
symbionts by Greek letters. The widely varying amounts of morphological data
available for each symbiont, and especially the paucity of biochemical data, make it
impossible for us to adopt a binomial system at present.
Symbionts which seem to form a group are given the same Greek letter. In addition,
each sample of a symbiont is characterized by the stock number of its host paramecium
(e.g. 51-kappa, 540-mu etc.), irrespective of whether two or more examples of a given
type of symbiont, occurring in different stocks, appear to be alike.
Kappa
Kappa is the original symbiont in P. aurelia discovered and named by Sonneborn
(1945). The most characteristic feature of kappa particles now appears to be their
5-2
68
G. H. Beak, A. Jurand and jf. R. Freer
content, at some stage in their development, of the peculiar structures called ' R '
(refractile) bodies (Figs, i, 2). There is usually one R body per kappa particle, but
occasionally two or even more may be seen (Preer & Stark, 1953). Killing activity of
kappa particles is associated with the presence of these R bodies (Preer, Siegel & Stark,
1953; Smith, 1961; Mueller, 1963; Preer & Preer, 1964). It is assumed that kappa
particles are released from paramecia into the medium, and if sensitive organisms take
up kappa particles which contain R bodies, killing may result.
Definition of kappa particles on the basis of their possession of R bodies is, however,
somewhat unsatisfactory. First, such particles (which were denoted 'brights' by
Preer & Stark, 1953) are never found alone in the cytoplasm of a paramecium. They
are always accompanied by a population of 'non-bright' kappa particles, the number
of which usually exceeds that of the 'brights'. It is known, moreover, that 'nonbrights' may develop R bodies and change into 'brights'. Furthermore, there are
some 'mutant' kappa particles which have lost the capacity to form 'brights', and no
longer have killing effects, though the paramecia bearing these mutant particles may
in some cases be immune to the killing action of the R bodies in other particles (Hanson,
1954; Widmayer, 1965; Mueller, 1964).
Thus kappa particles are those endosymbionts which either contain R bodies, or are
capable of differentiating into forms containing R bodies, or have been derived from
forms previously capable of developing R bodies. Furthermore, when kappa particles
kill, their killing action is always associated with R bodies. This rather confusing set
of criteria defining kappa would be avoided if the 'non-bright' forms of kappa
particles had sufficiently distinctive characters to provide a basis for a more satisfactory
definition. According to information available at this time, they do not do so.
It may be added that some mutant kappas incapable of forming R bodies have been
given a special symbol, pi (Hanson, 1954). We would, however, prefer to call such
particles kappa, since they differ from the original kappa to a relatively minor extent.
Indeed there may be a whole range of types, characterized by the frequency and
degree of development of R bodies occurring in a population of kappa particles
(see, for example, Widmayer, 1968).
Many different stocks of P. aurelia belonging to syngens 2 and 4, but so far to no
other syngen, have been found to contain kappa particles, as here defined. Indeed, in
Britain at least almost all wild populations of the very common syngen 2 seem to
contain kappa particles.
Variations of kappa are concerned with the detailed structure (size, shape, etc.) and
properties of the R bodies, the percentages of brights and non-brights, and the types
of pre-mortal effects such as spinning, hump formation, paralysis, etc. which have
been described by Sonneborn (1959). We describe and illustrate two distinct types:
those of syngen 2 (illustrated by two somewhat different strains, 7-kappa and 562kappa); and that of syngen 4 (51-kappa).
Syngen 2 kappa (7-kappa; 562-kappa). These two kappas are shown in Figs. 1-7.
The non-bright (N) forms are rod-shaped particles about 2 /i long, though in stock 562
some may reach 6 /<• or more in some instances (Fig. 6). Internally no marked features
are apparent. The outside of the particles is seen in some regions to consist of two
Endosymbionts of Paramecium
69
distinct membranes, each being a unit-membrane in the terminology of Robertson
(1959), comprising two electron-dense layers separated by a light one. The bright
(B) form of kappa is thicker, usually shorter, and more irregular in shape than the
N form. The outer unit membranes of bright and non-bright forms are indistinguishable. The R bodies in the syngen 2 kappas are plainly visible by phase-contrast
microscopy of squashes and, at the highest magnification, by dark or bright phasecontrast microscopy of paramecia stained by the osmium-lacto-orcein method (Figs.
1, 2). (Bright phase-contrast observation of R bodies in the osmium-lacto-orcein
method is considerable improved, and the appearance in dark phase contrast is not
harmed, by omission of the orcein from the stain.) In the electron microscope, the
R bodies are seen to be hollow membranous structures consisting of a long ribbon
wound into a tight spiral of about ten turns, as described previously (Anderson, Preer,
Preer & Bray, 1964; Preer, Hufnagel & Preer, 1966).
It has recently been shown that, associated with the R bodies of stocks 7 and 562,
and especially in their centres, one can see a group of polyhedral virus-like bodies, of
diameter approximately 500A (Preer & Preer, 1967; Preer & Jurand, 1968) (Figs. 3,
4, 14). Kappa particles lacking R bodies do not contain the virus-like particles. The
R bodies of stock 562 and stock 7 can be unwound by treatment with certain agents
and are then seen to consist of a tape-like structure with a rather blunt or irregularly
shaped outer end and a pointed inside end at which the virus-like material seems to be
concentrated. Unwinding occurs from the outside end.
562-Kappa and 7-kappa show a number of minor differences (L. B. Preer, personal
communication). The sheath-like structure (Fig. 3) surrounding the R bodies of stock 7
(Preer & Jurand, 1968) is absent in stock 562-kappa. Phosphotungstic acid, which
causes the R bodies of stock 7 to unroll, has little effect on the R bodies of stock 562.
The ribbon of the stock 562 R body is generally much narrower and shorter than it is
in stock 7. The numerous capsomere-like subunits found on the R bodies of stock 7
(Fig. 13) are not apparent in stock 562. Finally, 7-kappa induces sensitive strains of
paramecia to spin vigorously on their longitudinal axes, while 562-kappa induces
vacuolization prior to death. The range of variations amongst the many different
kappas of syngen 2 has not been investigated.
Syngen 4 kappa {51-kappa). The two forms of 51-kappa are shown in Fig. 8. Our
observations confirm in a general way those of Dippell (1958) and Rudenberg (1962).
These particles differ somewhat from those of 7-kappa in size and shape, the 51-kappa
particles being shorter—about 1-2 /t x 0-5-1 /i—(Sonneborn, 1961) than those of
7-kappa, and the 'brights' often being round or occasionally irregularly shaped
structures. Electron micrographs are shown in Figs. 10 and 11. The two membranes
are often quite distinct, with a tendency for the outer one to separate rather far from
the inner and simulate a vacuole. We suspect that such separation may be the result of
inadequate fixation.
The R bodies of stock 51 are generally similar in appearance to those of stock 7,
but differ in a number of details. The outside as well as the inside tips are acute (the
outside tip is blunt in stock 7). Unrolling occurs from the inside (rather than from the
outside as in stock 7) of the 51 R body and results in a very tightly wound hollow tube
70
G. H. Beale, A. Jurand and J. R. Freer
(rather than a loosely twisted ribbon). It was shown by Mueller (1962) that 51 -kappa R
bodies (unlike those of 7-kappa) unroll when exposed to acid. Preer et al. (1966) found
that lowering the pH to 6-o caused complete unrolling, but the unrolled ribbons rolled
up again if the pH was subsequently raised to 7-0.
It has been found that the R bodies of 51-kappa bear virus-like particles, which are,
however, quite distinct from those of 7-kappa. The 51-kappa 'viruses' are long helical
structures found within the original inside end of the unrolled tape (Preer & Preer,
1967).
We have noted that R bodies of stock 51-kappa particles, unlike those of syngen 2,
cannot be seen by phase-contrast microscopy of osmium-lacto-orcein stained preparations. It is likely that this phenomenon is a consequence of the fact that the acetic
acid (which is present in the stain) is strong enough to unroll the R bodies. In any
event, it is unfortunately not possible to see R bodies of syngen 4 kappa particles
in situ in the light microscope.
One of the 'mutants' of 51-kappa, 511T142, differs structurally from the abovedescribed standard type in having R bodies with shorter tapes but normal virus-like
elements (Widmayer, 1968), and one—51IT143—forms only non-bright forms (Fig. 9).
The other kappas of syngen 4 appear very similar to 51-kappa in the phase-contrast
microscope, but have not been investigated by electron microscopy.
Mu
Mu particles are defined as those endosymbionts which by their presence in a
paramecium make the latter a mate-killer. The phenomenon of mate-killing was
discovered by Siegel (1953, 1954), who found that following conjugation between
mate-killers and normal paramecia, ex-conjugants or later fission products deriving
cytoplasm from the normal parents were killed or damaged, whilst ex-conjugants
deriving cytoplasm (and mu particles) from mate-killer parents yielded viable progeny.
Three mate-killer stocks (arranged in a 'peck-order' of mating) were described in
syngen 8 (Siegel, 1953; Levine, 1953)- Subsequently mate-killers have been found in
syngen 1 (stocks 540, 548, 551, 555) (Beale 1957; Beale & Jurand, 1966, and unpublished observations) and in syngen 2 (stock 570, which we have now found brings
about mate-killing when allowed to conjugate with stock 562).
The type 540-mu has received most study. It is a rod-shaped particle, basically about
2 /t long, but under some conditions, such as starvation, gives very long forms, to
20 /i or more (Fig. 16). Mu particles are often arranged in clumps (Figs. 17, 18), and,
in the case of 540-mu, when placed on a glass slide in a drop of water adhere flat to the
glass. (Adsorption to glass is characteristic of a number of other particles, such as
7-kappa, but not of others, such as 51-kappa.)
In the electron microscope, two conspicuous outer membranes are seen, and in some
preparations a clear halo, which has been interpreted as a capsule (Beale & Jurand,
i960), surrounding the particles. Sometimes, however, the latter is not seen.
The internal contents of 540-mu particles do not show any noteworthy feature (Fig.
19). The other syngen 1 mus vary considerably in size, those of stock 548 being about
1 JLI in length, but appear generally to be similar to 540-mu. The syngen 8 mu particles
Endosymbionts of Paramecium
71
have not been studied by electron microscopy. Those of syngen 2 (stock 570) are
small (1 /() but apparently similar to the syngen 1 mu particles (Figs. 20, 21). Three
syngen 1 mu particles have been described in an earlier paper (Beale & Jurand, 1966).
Lambda and sigma
Paramecia containing lambda particles act as killers of the ' rapid lysis' type (Schneller, 1958, 1962). When mixed with sensitive paramecia the latter may be injured in
10 min and killed in 30 min (that is, much more rapidly than when kappa is the
killing particle). However, sensitivity to killing by lambda is restricted to certain
stocks of syngens 3, 5 and 9. Stocks of other syngens, though lacking lambda particles,
are resistant.
These symbionts are larger than kappa or mu. Lambda particles measure about
3 //, xo'5/i and resemble bacilli in general appearance (Fig. 22). They do not contain
R bodies or, so far as is known, any other special component associated with killing
activity. Jurand & Preer (1968) have recently made the surprising discovery that
lambda particles bear typical peritrichous bacterial flagella (Figs. 23, 24). Another
characteristic is the enclosure of the symbionts in cytoplasmic vacuoles, bounded by a
smooth membrane, one or a few symbionts lying in each vacuole (Fig. 24).
Lambda particles have been found in one stock of syngen 4 (stock 239) and several of
syngen 8 (stocks 216, 229, 299) (Sonneborn, Mueller & Schneller, 1959). Morphologically the particles in different stocks look alike, though there are minor differences in
size, numbers of flagella and density of particles within a paramecium.
Sigma is the name given to a type of symbiont occurring in a single stock (114) of
syngen 2 (Sonneborn et al. 1959). This is a very large particle (up to 15 /t long) and it
has an unusual sinuous shape (Fig. 25). In a number of respects, however, sigma
resembles lambda. Sigma bears flagella (Figs. 26, 27), is situated in vacuoles in the
cytoplasm of the paramecia, does not contain R bodies and is like lambda with regard
to rapidity and specificity of killing (Schneller, 1962). It would therefore be reasonable
to classify this particle with lambda.
Gamma
Two widely separated stocks of syngen 8 (214 from Florida, 565 from Uganda)
contain very small particles of apparently identical type (Figs. 28-31). The unit
particles are about 0-7 //, long, but are usually present as doublets. One of the most
striking features is the system of membranes surrounding the particles. Each gamma
particle, like other symbionts, is bounded by two membranes, but gamma is peculiar
in being enclosed also by a third membrane, to which ribosome-like particles are
attached. The third, outermost membrane may extend, at the ends of the gamma
particle, for some distance into the cytoplasm, and has the appearance of a piece of
endoplasmic reticulum.
No R bodies are present, but it was reported by Sonneborn (1956) that stock 214
secretes into the medium a material which causes sensitive paramecia to become spherical, greatly enlarged and die. We have observed that stock 565 paramecia have similar
72
G. H. Beak, A. Jurand and J. R. Freer
effects. In view of their distinctive morphology, we feel that these symbionts should be
given a special symbol—'gamma'.
Delta
This is a rod-shaped particle approximately 2 ft long, occasionally extending to
10/4 (Figs. 32-35). The main characteristic of delta is the layer of electron-dense
material surrounding the outer of the two membranes. This feature is indistinguishable
in the two examples found in stock 225 (syngen 6) and stock 561 (syngen 1). It is
because of this unique structure of the outer wall that we propose to group the two
types together and call them 'delta'. There is no membrane-bound vacuole as in the
case of lambda, or closely applied outer membrane as in the case of gamma. The electron
micrographs suggest that the particles in both stocks may occasionally bear sparse
flagella. 225-Delta has on a number of occasions (but not always) been found to be
motile in squashes; motility has not yet been observed in the case of 561-delta.
Stock 225 was shown by Sonneborn (1956) to be a weak paralysis killer; whether
stock 561 has killing properties is unknown.
Nu
Sonneborn et al. (1959) designated the particles in stocks 87 and 314 of syngen 5
as 'nu'. They have no known killing action. Holzman (1959) noted that particle-free
animals of these stocks were less resistant to the rapid-lysis killers than were animals
containing nus. We have found a number of other cytoplasmic particles, for example,
in stock 1010 (syngen 2) and stock Hu 35-1 (syngen 2), which are not known to cause
killing and which are not otherwise characterized in any special way. It is expedient
to group them all together, but they probably constitute a very heterogenous group,
apparently occurring in a number of syngens. Preliminary electron-microscope studies
of 87-nu and 1010-nu show them to possess small papilli attached to the membranes,
and embedded in capsules.
Alpha
These symbionts, which are at present known to occur in only a single stock (562)
of syngen 2 (in P. aurelid), differ from all those we have previously described in that
they are situated in the macronucleus. There are two rather distinct types (Figs. 36-41):
a short sickle-shaped form, about 2 /i long, occurring predominantly in actively growing
cultures of paramecia; and a longer, thin twisted form (about 6/t) with pointed ends,
occurring mainly in starving paramecia. A detailed account of these particles will be
published elsewhere (Preer, 1969).
Ordinarily alpha particles do not occur in the cytoplasm, though during the breakdown of the macronucleus at conjugation and autogamy some particles are liberated
into the cytoplasm, and may pass into the newly developing macronuclear Anlagen.
The symbionts do not occur in the micronuclei (Fig. 37). They readily pass from one
paramecium to another through the medium (Preer, 1969). No killing effect results
from the presence of these particles, so far as is known.
We have obtained evidence that for the maintenance of 562-alpha particles a
Endosymbionts of Paramecium
73
specific paramecium gene must be present. A cross was made between paramecia of
stocks 562 and 114 (which is unable to maintain alpha). Following autogamy of the
hybrid, an F2 was obtained comprising 26 clones capable of maintaining alpha (following
infection), and 29 clones unable to do so, indicating a 1:1 ratio, as would be expected
for the segregation of a pair of alleles.
Many years ago symbionts similar to alpha were described in a ciliate referred to as
'P. aurelia' (Petschenko, 1911) but from the description it appears to have been
Paramecium caudatum. (We have in our collections also a sample of P. caudatum
containing a different macronuclear symbiont). Petschenko named his particle
'Drepanospira miillerV.
It is also of interest to add here that the stock in which the alpha particles were
found (562) also contains kappa particles in the cytoplasm which have already been
mentioned above (p. 68).
DISCUSSION
From the above description it is obvious that the endosymbionts of P. aurelia
comprise a heterogeneous assortment of micro-organisms. In length they range from
°'5 /' (gamma) to 15 /,t (sigma, not including the exceptionally long forms of some
mu particles). They may lie freely in the cytoplasm or macronucleus, or be enclosed in
membrane-bound vacuoles of various types. The external membranes of the symbionts
show some variation, though two unit membranes are always present. All those
symbionts tested have been found to be Gram-negative: namely kappa (Preer & Stark,
1953), lambda (Soldo, 1963), mu (Stevenson, 1967a), and all the remainder, except that
gamma particles, when extracted from the paramecia, were found to show some
variability in their reaction to Gram's stain (C. N. Wiblin, unpublished observations).
Internally, not much detail is apparent by light or electron microscopy, apart from
the R bodies of kappa. In none of the symbionts so far studied is there a clearly
delimited nuclear region, such as is commonly found in free-living bacteria.
As regards toxic properties, different types of symbionts have different effects, and a
number have no obvious effect at all. The presence of substantial numbers of a
symbiont within a paramecium always protects that paramecium from killing by
exogenous symbiotic particles of the same type (Sonneborn, 1959).
Most observers have now accepted the view that the symbionts should be regarded as
bacteria (Preer & Stark, 1953; Dippell, 1958; Beale & Jurand, i960; Sonneborn, 1961;
Stevenson, 19676). Apart from size and morphology, there is a considerable amount of
evidence favouring this view. Both RNA and DNA have been shown to be present in
a number of particle types; for example, kappa (Preer, 1950; Dippell, 1959; SmithSonneborn & van Wagtendonk, 1964), mu (Beale & Jurand, i960; Stevenson, 1967a)
and lambda (van Wagtendonk & Tanguay, 1963). Some symbionts are sensitive to
antibiotics (see Sonneborn, 1959). Kappa particles have recently been shown by
Kung (1968) to contain many enzymes concerned with respiration, and mu particles
have been shown by Stevenson (1967 a) to contain a DNA-dependent RNA polymerase.
Stevenson (19676) showed that mu particles contain diaminopimelicacid, and possibly
74
G. H. Beak, A. Jurand and J. R. Freer
muramic acid, substances characteristic of bacterial cell walls. Van Wagtendonk,
Clark & Godoy (1963) reported that lambda particles could be cultivated in a paramecium-free medium. Kappa particles were shown by Sonneborn (1948) to be
transmissible, under special conditions, from cell to cell by infection. Preer (1968)
found that alpha particles are naturally infective from one paramecium to another.
However, in view of the pronounced morphological and other variations between
different types of particle, it would be unjustifiable to generalize from data obtained
from any one of them. We conclude that we are dealing with a miscellaneous collection
of bacteria which have come to occupy a specialized niche.
As regards the distribution of particular symbionts amongst different stocks and
syngens of paramecia, certain surprising features should be noted. Kappa particles,
as defined by possession of R bodies, occur only in syngens 2 and 4, but in those two
(especially syngen 2) kappa is found often. Moreover the R bodies seem to be of two
main types, one type being found in a number of stocks of syngen 2, another type
in syngen 4, notwithstanding the world-wide geographical range of each of these two
syngens. Sonneborn (1959) has pointed out that, amongst the syngen 4 killer stocks
containing kappa particles, seven appear to have identical killing actions (' humpkilling'), though the stocks come from widely scattered natural sources in North,
Central and South America and Japan.
Syngen 1, which is common in warm temperate regions all over the world, exhibits
relatively few examples of symbionts, but out of the five in our present collections
four are mus, though each of these is distinct judged both by morphology of the
particles and by their ability to make the host paramecia mate-killers of different types.
Another surprising 'coincidence' is the finding that two stocks of syngen 8, one
from Florida, the other from Uganda, bear almost identical symbionts of a highly
characteristic type (gamma). These are so far the only known occurrences of this
symbiont. In view of its potent killing properties, it is unlikely that other examples
would have been missed, had they been present in laboratory collections.
Finally, the delta particles of stock 561 (syngen 1) and stock 225 (syngen 6) are
morphologically almost indistinguishable in spite of their widely different origins.
Some syngens have not been found to contain symbionts at all. An example is
syngen 9, of which many Scottish stocks have been collected from the same ponds and
streams as syngen 2 stocks, which very frequently contain kappa particles.
Thus the distribution of the different types of symbiont amongst the stocks and
syngens of P. aurelia is markedly non-random. The implications of this are at
present not clear, but the following facts should be borne in mind.
We do not know how the different syngens of P. aurelia have evolved, or the
means by which they have arrived at their present wide, and in some cases world-wide
distribution (Sonneborn, 1957). Individual paramecia must remain in fresh water
at all times or they die. So far as we know, transport from one place to another is
accidental and probably rare. Most populations in enclosed waters are presumed to be
isolated. Even less do we know how the symbionts are spread about. One way, of
course, would be inside paramecia. To what extent any of the symbionts enjoy a freeliving existence is unknown, It is, however, known that paramecia which bear sym-
Endosymbionts of Paramecium
75
bionts readily lose them irreversibly. Re-infection must be exceedingly rare, since the
density of free-living symbionts in the vicinity of paramecia would be, at the very best,
exceedingly low.
There are several indications of the existence of specific adaptations between
symbionts and paramecia. For example, syngen 2 and syngen 9 are two common
syngens in Scotland and often found in the same sample of water. Syngen 2 stocks
nearly always contain symbionts; syngen 9 has never been found to contain them.
Secondly, there is the characteristic association of certain types of symbiont and
certain syngens, as mentioned above. Thirdly, there are many examples of symbionts
whose maintenance depends on the presence of a specific Paramecium gene. This
was shown first in the case of 51-kappa (Sonneborn, 1943), and subsequently for
various mu particles (Siegel, 1953; Gibson & Beale, 1961), lambda and gamma
(Sonneborn, et al. 1959) and alpha (as described above, p. 72).
In spite of the necessity for these supporting genes, and in spite of the high probability of a loss of symbionts (even when the genes are present), many wild stocks of
paramecia contain symbionts, as already mentioned. This raises the question of the
value of the symbiosis to either member of the partnership. The symbionts obviously
get a convenient and abundant supply of nutrients, and if capable of resisting digestion
by the enzymes of the paramecia, would appear to be in an advantageous and wellprotected environment. No obvious advantage to the paramecium has been demonstrated. Paramecia seem to grow equally well with or without symbionts. It is indeed
a striking fact that enormous numbers of 'foreign' micro-organisms, occupying an
appreciable proportion of the cell volume, may be present in the cytoplasm or nucleus,
without disturbing the life of the paramecium in any obvious way. Possibly some
symbionts aid their hosts by synthesizing some nutrient which the paramecia would
otherwise have to derive from the medium, and this could be so in the case of lambda
which, according to Soldo (1963) and van Wagtendonk et al. (1963), enables a paramecium to dispense with an external source of folic acid.
The killing properties of symbiont-bearing paramecia may not be of any significance
in nature, owing to the low densities of ciliates normally found. Killers and sensitives
are known to co-exist in the same small sample of water. On the other hand, paramecia
which contain symbionts are immune to the killing action of symbionts of the same
type, when present free in the water. Thus such paramecia would to some extent be
protected, though presumably against only a minute proportion of the possible toxic
micro-organisms in the environment.
Whatever their ecological significance, these symbiotic associations are very common
in P. aurelia and have also been reported in other ciliate protozoa. Sonneborn (1959)
cites a number of such examples, to which may now be added Euplotes minuta
(Heckmann, Preer & Straetling, 1967), and Tetrahymena sp., Halter ia grandinella and
Oxytricha bifara (van Wagtendonk & Soldo, 1965). Kirby (1941) describes a number
of examples of other protozoan symbionts. Since ciliates are continuously imbibing
bacteria-laden water, it might be thought that the environment of these organisms is
particularly favourable for the establishment of these symbioses. However, similar
phenomena may be quite common in other groups of animals. Buchner (1965) claims
76
G. H. Beak, A. Jurand and J. R. Freer
that over 10% of insects contain intracellular micro-organisms (see also Brooks, 1963).
Lanham (1968) has recently pointed out the similarities between some of these insect
symbionts (Blochmann bodies) and kappa or mu particles. Maillet & Folliot (1967)
have published an electron micrograph of a particle, remarkably similar to 540-mu,
in the spermatozoa of a homopterous insect, and in Drosophila, Yanders, Brewen, Peacock
& Goodchild, (1968) have described bacterium-like granules in the spermatocytes of
some strains. Finally, Woods & Bevan (1968) have described a killer factor in yeast.
If such occurrences are widespread, and if the symbionts have toxic effects, the
importance for cell-to-cell interactions would be considerable.
We wish to thank Dr D. Widmayer for kindly supplying the negative for Fig. 15.
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ABBREVIATIONS ON PLATES
al, alpha particle
B, bright kappa particle
caps, ' capsomeres'
cyt, cytoplasm
fl,
flagella
fv, food vacuole containing bacteria
Ib, ' large bodies (' nucleoli') in macronucleus
ma, macronucleus
mi, micronucleus
mu, mu particles
N, non-bright kappa particles
R, refractile body in bright particle
s, ' sheath' to R body
sb, ' small bodies' in macronucleus
v, virus-like particles in R bodies
vac, vacuole
The scale on Figures represents 1 /(. except where otherwise indicated.
Fig. 1. 7-kappa (syngen 2). Bright and non-bright particles. Osmium-lacto-orcein.
Dark phase-contrast, x 3000.
Fig. 2. 7-kappa, as in Fig. 1. Bright phase-contrast, x 3000.
Fig. 3. 7-kappa. Electron micrograph of longitudinal section through R body, x 60000.
Fig. 4. 7-kappa. Electron micrograph of transverse section through R body, x 60000.
Fig- 5- 7-kappa. Electron micrograph of section through non-bright (N) particle,
x 60000.
Endosymbionts of Paramecium
So
G. H. Beak, A. Jurand and J. R. Freer
Fig. 6. 562-kappa (syngen 2). Osmium-lacto-orcein. Dark phase-contrast, x 1200.
Fig. 7. 7-kappa (syngen 2). Osmium-lacto-orcein. Dark phase-contrast, x 1200.
Fig. 8. 51-kappa (syngen 4). Unfixed squash in bright phase-contrast, x 3000.
Fig. 9. 5im43-kappa (syngen 4). Unfixed squash in bright phase-contrast. Mutant
showing only N particles, x 3000.
Fig. 10. 51 -kappa (syngen 4). Electron micrograph showing transverse section through
R body, x 60000.
Fig. 11. 51-kappa (syngen 4). Electron micrograph showing longitudinal section through
R body, x 60000.
Endosymbionts of Paramecium
10*
81
G. H. Beale, A. Jurand and J. R. Preer
12
Fig.
Fig.
Fig.
Fig.
12. 7-kappa (syngen 2). Unwound R body x 14000.
13. 7-kappa (syngen 2). Unwound R body showing detail of inner end. x 140000.
14. 7-kappa (syngen 2). Virus-like particles, x 160000.
15. 51-kappa (syngen 4). Unwound R body showing helical virus-like particles.
x 120000.
Endosymbionts of Paramecium
83
20
Fijj;. 16. 540-nm (syn^cn 1). Starved animal showing short and long forms. O s m i u m lacto-orccin. Dark phase-contrast, x 1200.
Fig. 17. 54N-mu (syngen 1). f l u s t e r s of particles. Osmium-lacto-orcein. Dark p h a s e contrast, x 1200.
Fig. 18. 555-mu (syngen 1). Osmium-lacto-orcein. Dark phase contrast, x 1200.
Fig. 19. 540-mu (syngen 1). Electron micrograph, x 35000.
F'ig. 20. 570-mu (syngen 2). Electron micrograph, x 57000.
Fig. 2 1 . 548-mu (syngen 1). Electron micrograph, x 60000.
(.-2
84
G. II. Beak, A. Jurand and J. R. Preer
Fig. 22. 239-lambda (syngen 4). Osmium-lacto-orcein. Dark phase-contrast, x 1200.
Fig. 23. 239-lambda (syngen 4). Negatively stained particle showing Hagella. x 19500.
Fig. 24. 216-lambda (syngen 8). Electron micrograph of section showing particle and
flagella in vacuole. x 36000.
Endosvmbionts of Paramecium
Fig. 25. 114-sigma
Fig. 26. 114-sigma
flagella in vacuole.
Fig. 27. 114-sigma
(svngen 2). Osmium-lacto-orcein. Dark phase-contrast, x 1200.
(svngen 2). Electron micrograph of section showing particle and
x 54000.
(svngen 2). Negatively stained particle showing flagella. x 17000.
86
G. H. Beak, A. jfurand and J. R. Preer
Fig. 28. 214-gamma (syngen 8). Osmium-lacto-orcein. Dark phase-contrast, x 1200.
Fig. 29. 565-gamma (syngen 8). Osmium-lacto-orcein. Dark phase-contrast, x 1200.
Fig. 30. 214-gamma (syngen 8). Electron micrograph, x 60000.
Fig- 3 1 - 565-gamma (syngen 8). Electron micrograph. Note outermost membrane
trailing off into cytoplasm (arrow), x 60000.
Endosymbionts of Paramecium
G. H. Beak, A. Jurand and J. R. Preer
Fig. 32. 225-delta (syngen 6). Osmium-lacto-orcein. Dark phase-contrast. Long and
short forms, x 1200.
Fig- 33- 561-delta (syngen 1). Osmium-lacto-orcein. Dark phase-contrast, x 1200.
Fig. 34. 225-delta (syngen 6). Electron micrograph. Note the electron-dense material
on the outer membrane (arrow), x 57000.
Fig- 35- 561-delta (syngen 1). Electron micrograph, x 56000.
Endosymbionts of Paramecium
\
^
* *>i!
its'
90
G. H. Beak, A. Jurand and J. R. Freer
Fig. 36. 562-alpha (syngen 2). Sickle-shaped particles in macronucleus of growing
paramecium. Osmium-lacto-orcein. Dark phase-contrast, x 1200.
Fig. 37. 562-alpha (syngen z). Slender forms in macronucleus of starving paramecium.
Osmium-lacto-orcein. Dark phase-contrast, x 1200.
Fig. 38. 562-alpha (syngen 2). Isolated particles in bright phase-contrast, x 3000.
Fig. 39. 562-alpha (syngen 2). Electron micrograph of a section of sickle-shaped particle,
x 39000.
Fig. 40. 562-alpha (syngen 2). Electron micrograph of a section through the macronucleus. x 11000.
Fig. 41. 562-alpha (syngen 2). Electron micrograph of a section of a long slender
form, x 60000.
Endosymbionts of Paramecium