J. Cell Sci. 5, 65-91 (1969) 65 Printed in Great Britain

THE CLASSES OF ENDOSYMBIONT OF AURELIA

G. H. BEALE AND A. JURAND Institute of Animal , 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 Gram- negative 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 . 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 . 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 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 dis- covery by Sonneborn (1938a), that certain strains of the 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 66 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 'rapid- lysis' 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 know- ledge 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 micro- scopy 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 Type of symbiont

54° Mexico Mu 548 Los Angeles, California, U.S.A. Mu 55i San Francisco, California, U.S.A. Mu 555 Monterey, California, U.S.A. Mu 561 Pisa, Italy Delta 2 7 N. Carolina, U.S.A. Kappa 562 Milan, Italy Kappa and alpha 57O Georgia, U.S.S.R. Mu 114 Bloomington, Indiana, U.S.A. Sigma IOIO Cove Lake, Tennessee, U.S.A. Nu Hu 35-1 Edinburgh, Scotland Nu 4 51 Spencer, Indiana, U.S.A. Kappa 239 Holmes Co., Florida, U.S.A. Lambda 5 87 Philadelphia, U.S.A. Nu 314 Pike Co., Illinois, U.S.A. Nu 6 225 Florida, U.S.A. Delta 8 216 Florida, U.S.A. Lambda 229 Florida, U.S.A. Lambda 299 Panama Lambda 214 Florida, U.S.A. Gamma 565 Uganda 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 'non- brights' 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 562- kappa); 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 indistinguish- able. 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 phase- contrast 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 . 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 prepara- tions. 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 above- described 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 unpub- lished 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 (Schnel- ler, 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). Morphologi- cally 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 spheri- cal, 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 break- down 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; Smith- Sonneborn & 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 para- mecium-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 (' hump- killing'), 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 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 free- living 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 prob- ability 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 well- protected environment. No obvious advantage to the paramecium has been demon- strated. 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 para- mecium 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 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.

REFERENCES ANDERSON, T. F., PREER, J. R., PREER, L. B. & BRAY, M. (1964). Studies on killing particles from Paramecium: the structure of refractile bodies from kappa particles. J. Microscopie 3, 395-402. BEALE, G. H. (1957). A mate-killing strain of Paramecium aurelia, variety 1, from Mexico. Proc. R. phys. Soc. Edinb. 26, 11-14. BEALE, G. H. & JURAND, A. (i960). Structure of the mate-killer (mu) particles in Paramecium aurelia, stock 540. J. gen. Microbiol 23, 243-252. BEALE, G. H. & JURAND, A. (1966). Three different types of mate-killer (mu) particle in Paramecium aurelia (syngen i).J. Cell Sci. I, 31-34. BROOKS, M. A. (1963). Symbiosis and aposymbiosis in Arthropods. Symp. Soc. gen. Microbiol. 13, 200-231. BUCHNER, P. (1965). Endosymbiosis of Animals with Plant. Microorganisms, pp. 1-909. New York: Interscience (Wiley). DIPPELL, R. V. (1950). Mutation of the killer cytoplasmic factor in Paramecium aurelia. Heredity, Lond. 4, 165-188. DIPPELL, R. (1958). The fine structure of kappa in killer stock 51 of Paramecium aurelia. J. biophys. biochem. Cytol. 4, 125-128. DIPPELL, R. V. (1959). The distribution of DNA in kappa particles of Paramecium in relation to the problem of their bacterial affinities. Science, N.Y. 130, 1415. GIBSON, I. & BEALE, G. H. (1961). Genie basis of the mate-killer trait in Paramecium aurelia, stock 540. Genet. Res. 2, 82-91. HANSON, E. D. (1954). Studies on kappa-like particles in sensitives of Paramecium aurelia, variety 4. Genetics, Princeton 39, 229-239. HECKMANN, K., PREER, J. R. & STRAETLING, W. H. (1967). Cytoplasmic particles in the killers of Euplotes minuta and their relationship to the killer substance. J. Protozool. 14, 360-363. HOLZMAN, H. E. (1959). A kappa-like particle in a non-killer stock of Paramecium aurelia, syngen 5. J. Protozool. 6 (suppl.), 26. JURAND, A. & PREER, L. B. (1968). Ultrastructure of flagellated lambda symbionts in Para- mecium aurelia. J. gen. Microbiol 54, 359-364. KIRBY, H. (1941). Organisms living on and in Protozoa. In Protozoa in Biological Research (ed. G. N. Calkins & F. M. Summers), chapter 20, pp. 1009-1113. New York: Columbia University Press. KUNG, C. (1968). Oxidative Metabolism of Kappa Particles from Paramecium aurelia, Stock 51 in Relation to Their Nature and Origin. Ph.D Thesis, University of Philadelphia. LANHAM, U. N. (1968). The Blochmann bodies: hereditary intracellular symbionts of insects. Biol. Rev. 43, 269-286. LEVINE, M. (1953). The diverse mate-killers of Paramecium aurelia, variety 8: their inter- relations and genetic basis. Genetics, Princeton 38, 561-578. MAILLET, P. L. & FOLLIOT, R. (1967). Nouvelles observations sur le transport de micro- organismes intranucleaires appelds particules phi par les spermatozoides chez des insectes homopteres. C. r. liebd. Se'anc. Acad. Sci., Paris 264, 695—69S. Endosymbionts of Paramecium 77 MUELLER, J. A. (1962). Induced physiological and morphological changes in the B particle and R body from killer paramecia. J. Protozool. 9, 26. MUELLER, J. A. (1963). Separation of kappa particles with infective activity from those with killing activity and identification of the infective particles in Paramecium anrelia. Expl Cell Res. 30, 492-508. MUELLER, V. A. (1964). Paramecia develop immunity against kappa. Am. Zool. 4, 313-314. PETSCHENKO, B. (191 I). Drepanospira Muelleri n.g.n. sp-parasite des ; contribu- tion a l'etude de la structure des bacteYies. Arch. Protistenk. 22, 252-298. PREER, J. R. (1950). Microscopically visible bodies in the cytoplasm of the 'killer' strains of Paramecium anrelia. Genetics, Princeton 35, 344-362. PREER, J. R., HUFNAGEL, L. A. & PREER, L. B. (1966). Structure and behavior of R bodies from killer paramecia. J. Ultrastruct. Res. 15, 131-143. PREER, J. R. & JURAND, A. (1968). The relation between virus-like particles and R bodies of Paramecium anrelia. Genet. Res. 12, 331-340. PREER, L. B. & PREER, J. R. (1964). Killing activity from lysed particles of Paramecium. Genet. Res. 5, 230-239. PREER, J. R. & PREER, L. B. (1967). Virus-like bodies in killer paramecia. Proc. natn. Acad. Sci. U.S.A. 58, 1774-1781. PREER, J. R. & STARK, P. (1953). Cytological observations on the cytoplasmic factor 'kappa' in Paramecium aurelia. Expl Cell Res. 5, 478-491. PREER, J. R., SIEGEL, R. W. & STARK, P. S. (1953). The relationship between kappa and para- mecia in Paramecium aurelia. Proc. natn. Acad. Sci. U.S.A. 39, 1228-1233. PREER, L. B. (1969). Alpha, an infectious macronuclear symbiont of Paramecium aurelia. J. Protozool. 16, (in the Press). ROBERTSON, J. D. (1959). The ultrastructure of cell membranes and their derivatives. In The Structure and Function of Subcellular Components. Biochem. Soc. Symp. No. 16, (ed. E. M. Crook), pp. 3-43. Cambridge: University Press. RUDENBERG, F. H. (1962). Electron microscopic observations of kappa in Paramecium aurelia. Tex. Rep. Biol. Med. 20, 105-112. SCHNELLER, M. V. (1958). A new type of killing action in a stock of Paramecium aurelia from Panama. Proc. Indian. Acad. Sci. 67, 302. SCHNELLER, M. V. (1962). Some notes on the rapid lysis type of killing found in Paramecium aurelia. Am. Zool. 2, 446. SIECEL, R. W. (1953). A genetic analysis of the mate-killer trait in Paramecium aurelia, variety 8. Genetics, Princeton 38, 550-560. SIEGEL, R. W. (1954). Mate-killing in Paramecium aurelia variety 8. Physiol. Zool. 27, 89-100. SMITH, J. E. (1961). Purification of kappa particles of Paramecium aurelia, stock 51. Am. Zool. 1, 39°- SMITH-SONNEBORN, J. E. & VAN WAGTENDONK, W. J. (1964). Purification and chemical character- ization of kappa of stock 51 Paramecium aurelia. Expl Cell Res. 33, 50-59. SOLDO, A. T. (1963). Axenic culture of Paramecium. Some observations on the growth behaviour and nutritional requirements of a particle-bearing strain of Paramecium aurelia 299 lambda. Ann. N.Y. Acad. Sci. 108, 380-388. SONNEBORN, T. M. (1938a). Mating types, toxic interactions and heredity in Paramecium aurelia. Science, N.Y. 88, 503. SONNEBORN, T. M. (19386). Mating types in Paramecium aurelia: diverse conditions for mating in different stocks; occurrence, number and interrelations of the types. Proc. Am.phil. Soc. 79, 411-434. SONNEBORN, T. M. (1943). Gene and cytoplasm. I. The determination and inheritance of the killer character in variety 4 of Paramecium aurelia. Proc. natn. Acad. Sci. U.S.A. 29, 320-343- SONNEBORN, T. M. (1945). The dependence of the physiological action of a gene on a primer and the relation of primer to gene. Am. Nat. 49, 318—339. SONNEBORN, T. M. (1948). Symposium on plasmagenes, genes and characters in Paramecium aurelia. Am. Nat. 82, 26-34. SONNEBORN, T. M. (1950). Methods in the general biology and genetics of Paramecium aurelia. J. exp. Zool. 113, 87-143. 78 G. H. Beak, A. Jurand and J. R. Freer SONNEBORN, T. M. (1956). The distribution of killers among the varieties of Paramecium aurelia. Anat. Rec. 125, 567-568. SONNEBORN, T. M. (1957). Breeding systems, reproductive methods, and species problems in Protozoa. In The Species Problem (ed. E. Mayr). Publs Am. Ass. Advvit Sci. 50, 155-324. SONNEBORN, T. M. (1959). Kappa and related particles in Paramecium. Adv. Virus Res. 6, 229-356. SONNEBORN, T. M. (1961). Kappa particles and their bearing on host-parasite relations. In Perspectives in Virology, vol. 2 (ed. M. Pollard), pp. 5-12. Minneapolis: Burgess. SONNEBORN, T. M., MUELLER, J. A. & SCHNELLER, M. V. (1959). The classes of kappa-like particles in Paramecium aurelia. Anat. Rec. 134, 642. STEVENSON, I. (1967a). Genetic and Biochemical Studies on Cytoplasmic Particles in Paramecium. Ph.D. Thesis, University of Edinburgh. STEVENSON, I. (19676). Diaminopimelic acid in the mu particles of Paramecium aurelia. Nature, Lond. 215, 434-435. WAGTENDONK, W. J. VAN, CLARK, J. A. D. & GODOY, G. A. (1963). The biological status of lambda and related particles in Paramecium aurelia. Proc. natn. Acad. Sci. U.S.A. 50, 835-838. WAGTENDONK, W. J. VAN & SOLDO, A. T. (1965). Endosymbiotes of ciliated protozoa. In Progress in Protozoology. Excerpta Medica Foundation. International Congress Series. 91, 244-245- WAGTENDONK, W. J. VAN & TANGUAY, R. B. (1963). The chemical composition of lambda in Paramecium aurelia, stock 299. J. gen. Microbiol. 33, 395-400. WIDMAYER, D. J. (1965). A non-killer resistant kappa and its bearing on the interpretation of kappa in Paramecium aurelia. Genetics, Princeton 51, 613-623. WIDMAYER, D. J. (1968). Abnormal refractile bodies in mutant kappa of Paramecium aurelia. Proc. XII int. Congr. Genetics, vol. 1, 71 (Abstr.). WOODS, D. R. & BEVAN, E. A. (1968). Studies on the nature of the killer factor produced by Saccharomyces cerevisiae. jf. gen. Microbiol. 51, 115-126. YANDERS, A. F., BREWEN, J. G., PEACOCK, W. J. & GOODCHILD, D. J. (1968). Meiotic drive and visible polarity in Drosophila spermatocytes. Genetics, Princeton 59, 245-253.

(Received 11 October 1968) ABBREVIATIONS ON PLATES al, alpha particle mi, micronucleus B, bright kappa particle mu, mu particles caps, ' capsomeres' N, non-bright kappa particles cyt, cytoplasm R, refractile body in bright particle fl, flagella s, ' sheath' to R body fv, food vacuole containing bacteria sb, ' small bodies' in macronucleus Ib, ' large bodies (' nucleoli') in macronucleus v, virus-like particles in R bodies ma, macronucleus 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 81

10* G. H. Beale, A. Jurand and J. R. Preer

12 Fig. 12. 7-kappa (syngen 2). Unwound R body x 14000. Fig. 13. 7-kappa (syngen 2). Unwound R body showing detail of inner end. x 140000. Fig. 14. 7-kappa (syngen 2). Virus-like particles, x 160000. Fig. 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. Osmium- lacto-orccin. Dark phase-contrast, x 1200. Fig. 17. 54N-mu (syngen 1). flusters of particles. Osmium-lacto-orcein. Dark phase- 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. 21. 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 (svngen 2). Osmium-lacto-orcein. Dark phase-contrast, x 1200. Fig. 26. 114-sigma (svngen 2). Electron micrograph of section showing particle and flagella in vacuole. x 54000. Fig. 27. 114-sigma (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- 31- 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 macro- nucleus. x 11000. Fig. 41. 562-alpha (syngen 2). Electron micrograph of a section of a long slender form, x 60000. Endosymbionts of Paramecium