THE CLASSES OF ENDOSYMBIONT OF PARAMECIUM AURELIA

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

THE CLASSES OF ENDOSYMBIONT OF

PARAMECIUM AURELIA

G. H. BEALE AND A. JURANDInstitute of Animal Genetics, Edinburgh 9, Scotland

AND J. R. PREERDepartment of Zoology, Indiana University, Bloomington, Indiana 47401, U.S.A.

SUMMARYThe endosymbionts of Paramecium aurelia appear to consist of a number of different Gram-

negative bacteria which have come to live within many strains of paramecia. It is not knownwhether in nature this relationship is mutually beneficial or not. The symbionts from oneparamecium may kill other paramecia lacking that kind of symbiont. We identify the followingclasses 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 theproduction of a toxin which kills sensitive paramecia. In certain mutants of kappa found in thelaboratory both R bodies and ability to kill have been lost. Second, mu particles (in syngens i, 2and 8) produce the phenomenon of mate-killing. Third, lambda (syngens 4 and 8) and sigmaparticles (syngen 2) are very large, flagellated organisms which kill only paramecia of syngens3, 5 and 9, and are enclosed in membrane-bound vacuoles. Fourth, gamma particles (syngen 8)are minute endosymbionts, surrounded by an additional membrane resembling endoplasmicreticulum. They have strong killing activity but no R bodies. Fifth, delta particles (syngens 1and 6) possess a dense layer covering the outer membrane. At least one of the two known stocksis a killer. Sixth, nu particles are a heterogeneous group of particles (syngens 2 and 5) which donot 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 ofthe 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 whichgrow in the cytoplasm or macronucleus of Paramecium aurelia. In using the word'endosymbiont', we simply mean one organism living within the cells of another, anddo not imply anything about the interactions between host and symbiont, whetherbeneficial or harmful. Knowledge of the symbionts of P. aurelia stems from the dis-covery by Sonneborn (1938a), that certain strains of the ciliate are 'killers'. Thekiller paramecia were originally inferred to contain kappa particles in the cytoplasmfrom the inheritance of the killer trait (Sonneborn, 1945). It was shown that, due tothe presence of kappa, a toxic material, at first called 'paramecin', was released intothe water, and sensitive paramecia in the vicinity were damaged or killed.

Later, kappa particles were identified under the light microscope by examination offixed and stained paramecia (Preer, 1950) and by observations with the phase-contrastmicroscope 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 asillustrations of non-Mendelian heredity. However, in the course of time attention hasbeen concentrated more on the nature of the particles themselves. By 1959 suchdetails of the structure and composition of kappa (and related) particles as were thenknown, showed that they were as large and complex as bacteria, though kappa particleshad 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 containdistinct cytoplasmic particles, those in mate-killers being called 'mu', and those inrapid-lysis killers 'lambda'. Moreover, a number of variants (or 'mutants') of theoriginal kappa particles were found (Dippell, 1950). One variant discovered byHanson (1954) was denoted 'pi'. These pi particles were distinguished from theoriginal 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 meansuncommon constitutents of paramecia. Sonneborn (1956) estimated that at least 30%of stocks of certain syngens of P. aurelia when first collected from nature were killersor mate-killers.

It has been known for many years that if paramecia are washed in bacteria-freemedium, crushed and observed in the phase-contrast microscope the presence ofendosymbionts may be ascertained, irrespective of whether there is any killing effector not (Preer & Stark, 1953). More recently a simple and much more rapid techniquehas been devised whereby one can quickly see whether kappa or other particles arepresent in a paramecium (Beale & Jurand, 1966). It is now known that a substantialproportion of wild strains of paramecia contain symbionts in the cytoplasm, and someeven in the macronucleus. So many types have now come to light that we feel it isdesirable to attempt a comparative survey and an assessment of the homologies andsignificance 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) detailsof the methods used in collecting and cultivating paramecia are given by Sonneborn(1950); (2) the demonstration of killing (including mate-killing phenomena) isdescribed by Sonneborn (1959); (3) for recognition of endosymbionts by light micro-scopy of stained whole paramecia see Beale & Jurand (1966) and by phase-contrastmicroscopy of crushed paramecia see Preer & Stark (1953); (4) for techniques forelectron microscopy see Jurand & Preer (1968).

The material used in the various studies referred to in this paper belongs to a numberof 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 stockbeing the progeny of a single individual collected from nature. Although in thisaccount 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 syngensappear 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

2

4

5

6

8

Stock no.

54°54855i555561

756257O1 1 4

I O I O

Hu 35-1

51

2 3 9

873 1 4

2 2 5

2 1 6

2 2 9

2 9 9

2 1 4

565

Place collected

MexicoLos Angeles, California, U.S.A.San Francisco, California, U.S.A.Monterey, California, U.S.A.Pisa, ItalyN. Carolina, U.S.A.Milan, ItalyGeorgia, U.S.S.R.Bloomington, Indiana, U.S.A.Cove Lake, Tennessee, U.S.A.Edinburgh, ScotlandSpencer, 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.PanamaFlorida, U.S.A.Uganda

Type of symbiont

MuMuMuMuDeltaKappaKappa and alphaMuSigmaN uNu

KappaLambdaNuNu

DeltaLambdaLambdaLambdaGammaGamma

THE CLASSES OF SYMBIONTS

Introduction

In this survey we shall continue to use the system of denoting the principal types ofsymbionts by Greek letters. The widely varying amounts of morphological dataavailable for each symbiont, and especially the paucity of biochemical data, make itimpossible 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 giventype 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, butoccasionally two or even more may be seen (Preer & Stark, 1953). Killing activity ofkappa 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 kappaparticles are released from paramecia into the medium, and if sensitive organisms takeup 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' byPreer & Stark, 1953) are never found alone in the cytoplasm of a paramecium. Theyare always accompanied by a population of 'non-bright' kappa particles, the numberof which usually exceeds that of the 'brights'. It is known, moreover, that 'non-brights' may develop R bodies and change into 'brights'. Furthermore, there aresome 'mutant' kappa particles which have lost the capacity to form 'brights', and nolonger have killing effects, though the paramecia bearing these mutant particles mayin 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 arecapable of differentiating into forms containing R bodies, or have been derived fromforms previously capable of developing R bodies. Furthermore, when kappa particleskill, their killing action is always associated with R bodies. This rather confusing setof criteria defining kappa would be avoided if the 'non-bright' forms of kappaparticles had sufficiently distinctive characters to provide a basis for a more satisfactorydefinition. 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 beengiven a special symbol, pi (Hanson, 1954). We would, however, prefer to call suchparticles 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 anddegree 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 noother syngen, have been found to contain kappa particles, as here defined. Indeed, inBritain at least almost all wild populations of the very common syngen 2 seem tocontain kappa particles.

Variations of kappa are concerned with the detailed structure (size, shape, etc.) andproperties of the R bodies, the percentages of brights and non-brights, and the typesof pre-mortal effects such as spinning, hump formation, paralysis, etc. which havebeen 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 562some may reach 6 /<• or more in some instances (Fig. 6). Internally no marked featuresare apparent. The outside of the particles is seen in some regions to consist of two


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 theN 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-contrastmicroscopy 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-orceinmethod is considerable improved, and the appearance in dark phase contrast is notharmed, by omission of the orcein from the stain.) In the electron microscope, theR bodies are seen to be hollow membranous structures consisting of a long ribbonwound 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, ofdiameter approximately 500A (Preer & Preer, 1967; Preer & Jurand, 1968) (Figs. 3,4, 14). Kappa particles lacking R bodies do not contain the virus-like particles. TheR bodies of stock 562 and stock 7 can be unwound by treatment with certain agentsand are then seen to consist of a tape-like structure with a rather blunt or irregularlyshaped outer end and a pointed inside end at which the virus-like material seems to beconcentrated. Unwinding occurs from the outside end.

562-Kappa and 7-kappa show a number of minor differences (L. B. Preer, personalcommunication). The sheath-like structure (Fig. 3) surrounding the R bodies of stock 7(Preer & Jurand, 1968) is absent in stock 562-kappa. Phosphotungstic acid, whichcauses 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 isin 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 ofparamecia to spin vigorously on their longitudinal axes, while 562-kappa inducesvacuolization prior to death. The range of variations amongst the many differentkappas of syngen 2 has not been investigated.

Syngen 4 kappa {51-kappa). The two forms of 51-kappa are shown in Fig. 8. Ourobservations 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-kappaparticles being shorter—about 1-2 /t x 0-5-1 /i—(Sonneborn, 1961) than those of7-kappa, and the 'brights' often being round or occasionally irregularly shapedstructures. Electron micrographs are shown in Figs. 10 and 11. The two membranesare often quite distinct, with a tendency for the outer one to separate rather far fromthe inner and simulate a vacuole. We suspect that such separation may be the result ofinadequate 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 (theoutside tip is blunt in stock 7). Unrolling occurs from the inside (rather than from theoutside 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 Rbodies (unlike those of 7-kappa) unroll when exposed to acid. Preer et al. (1966) foundthat lowering the pH to 6-o caused complete unrolling, but the unrolled ribbons rolledup 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 helicalstructures 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 aceticacid (which is present in the stain) is strong enough to unroll the R bodies. In anyevent, it is unfortunately not possible to see R bodies of syngen 4 kappa particlesin 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-likeelements (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-contrastmicroscope, but have not been investigated by electron microscopy.

Mu

Mu particles are defined as those endosymbionts which by their presence in aparamecium make the latter a mate-killer. The phenomenon of mate-killing wasdiscovered by Siegel (1953, 1954), who found that following conjugation betweenmate-killers and normal paramecia, ex-conjugants or later fission products derivingcytoplasm from the normal parents were killed or damaged, whilst ex-conjugantsderiving cytoplasm (and mu particles) from mate-killer parents yielded viable progeny.

Three mate-killer stocks (arranged in a 'peck-order' of mating) were described insyngen 8 (Siegel, 1953; Levine, 1953)- Subsequently mate-killers have been found insyngen 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 bringsabout mate-killing when allowed to conjugate with stock 562).

The type 540-mu has received most study. It is a rod-shaped particle, basically about2 /t long, but under some conditions, such as starvation, gives very long forms, to20 /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 theglass. (Adsorption to glass is characteristic of a number of other particles, such as7-kappa, but not of others, such as 51-kappa.)

In the electron microscope, two conspicuous outer membranes are seen, and in somepreparations 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 about1 JLI in length, but appear generally to be similar to 540-mu. The syngen 8 mu particles


have not been studied by electron microscopy. Those of syngen 2 (stock 570) aresmall (1 /() but apparently similar to the syngen 1 mu particles (Figs. 20, 21). Threesyngen 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 in10 min and killed in 30 min (that is, much more rapidly than when kappa is thekilling particle). However, sensitivity to killing by lambda is restricted to certainstocks 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 about3 //, xo'5/i and resemble bacilli in general appearance (Fig. 22). They do not containR bodies or, so far as is known, any other special component associated with killingactivity. Jurand & Preer (1968) have recently made the surprising discovery thatlambda particles bear typical peritrichous bacterial flagella (Figs. 23, 24). Anothercharacteristic is the enclosure of the symbionts in cytoplasmic vacuoles, bounded by asmooth 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 ofsyngen 8 (stocks 216, 229, 299) (Sonneborn, Mueller & Schneller, 1959). Morphologi-cally the particles in different stocks look alike, though there are minor differences insize, 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) ofsyngen 2 (Sonneborn et al. 1959). This is a very large particle (up to 15 /t long) and ithas an unusual sinuous shape (Fig. 25). In a number of respects, however, sigmaresembles lambda. Sigma bears flagella (Figs. 26, 27), is situated in vacuoles in thecytoplasm of the paramecia, does not contain R bodies and is like lambda with regardto rapidity and specificity of killing (Schneller, 1962). It would therefore be reasonableto 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 unitparticles are about 0-7 //, long, but are usually present as doublets. One of the moststriking features is the system of membranes surrounding the particles. Each gammaparticle, like other symbionts, is bounded by two membranes, but gamma is peculiarin being enclosed also by a third membrane, to which ribosome-like particles areattached. The third, outermost membrane may extend, at the ends of the gammaparticle, for some distance into the cytoplasm, and has the appearance of a piece ofendoplasmic reticulum.

No R bodies are present, but it was reported by Sonneborn (1956) that stock 214secretes 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


effects. In view of their distinctive morphology, we feel that these symbionts should begiven a special symbol—'gamma'.

Delta

This is a rod-shaped particle approximately 2 ft long, occasionally extending to10/4 (Figs. 32-35). The main characteristic of delta is the layer of electron-densematerial surrounding the outer of the two membranes. This feature is indistinguishablein the two examples found in stock 225 (syngen 6) and stock 561 (syngen 1). It isbecause of this unique structure of the outer wall that we propose to group the twotypes together and call them 'delta'. There is no membrane-bound vacuole as in thecase of lambda, or closely applied outer membrane as in the case of gamma. The electronmicrographs suggest that the particles in both stocks may occasionally bear sparseflagella. 225-Delta has on a number of occasions (but not always) been found to bemotile 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; whetherstock 561 has killing properties is unknown.

Nu

Sonneborn et al. (1959) designated the particles in stocks 87 and 314 of syngen 5as 'nu'. They have no known killing action. Holzman (1959) noted that particle-freeanimals of these stocks were less resistant to the rapid-lysis killers than were animalscontaining 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 causekilling and which are not otherwise characterized in any special way. It is expedientto group them all together, but they probably constitute a very heterogenous group,apparently occurring in a number of syngens. Preliminary electron-microscope studiesof 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 thatthey 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 growingcultures 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 bepublished 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 liberatedinto 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 oneparamecium to another through the medium (Preer, 1969). No killing effect resultsfrom the presence of these particles, so far as is known.

We have obtained evidence that for the maintenance of 562-alpha particles a


specific paramecium gene must be present. A cross was made between paramecia ofstocks 562 and 114 (which is unable to maintain alpha). Following autogamy of thehybrid, an F2 was obtained comprising 26 clones capable of maintaining alpha (followinginfection), and 29 clones unable to do so, indicating a 1:1 ratio, as would be expectedfor 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 beenParamecium caudatum. (We have in our collections also a sample of P. caudatumcontaining 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 werefound (562) also contains kappa particles in the cytoplasm which have already beenmentioned above (p. 68).

DISCUSSION

From the above description it is obvious that the endosymbionts of P. aureliacomprise a heterogeneous assortment of micro-organisms. In length they range from°'5 /' (gamma) to 15 /,t (sigma, not including the exceptionally long forms of somemu particles). They may lie freely in the cytoplasm or macronucleus, or be enclosed inmembrane-bound vacuoles of various types. The external membranes of the symbiontsshow some variation, though two unit membranes are always present. All thosesymbionts tested have been found to be Gram-negative: namely kappa (Preer & Stark,1953), lambda (Soldo, 1963), mu (Stevenson, 1967a), and all the remainder, except thatgamma particles, when extracted from the paramecia, were found to show somevariability in their reaction to Gram's stain (C. N. Wiblin, unpublished observations).

Internally, not much detail is apparent by light or electron microscopy, apart fromthe R bodies of kappa. In none of the symbionts so far studied is there a clearlydelimited nuclear region, such as is commonly found in free-living bacteria.

As regards toxic properties, different types of symbionts have different effects, and anumber have no obvious effect at all. The presence of substantial numbers of asymbiont within a paramecium always protects that paramecium from killing byexogenous symbiotic particles of the same type (Sonneborn, 1959).

Most observers have now accepted the view that the symbionts should be regarded asbacteria (Preer & Stark, 1953; Dippell, 1958; Beale & Jurand, i960; Sonneborn, 1961;Stevenson, 19676). Apart from size and morphology, there is a considerable amount ofevidence favouring this view. Both RNA and DNA have been shown to be present ina 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 toantibiotics (see Sonneborn, 1959). Kappa particles have recently been shown byKung (1968) to contain many enzymes concerned with respiration, and mu particleshave been shown by Stevenson (1967 a) to contain a DNA-dependent RNA polymerase.Stevenson (19676) showed that mu particles contain diaminopimelicacid, and possibly


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 betransmissible, 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 betweendifferent types of particle, it would be unjustifiable to generalize from data obtainedfrom any one of them. We conclude that we are dealing with a miscellaneous collectionof bacteria which have come to occupy a specialized niche.

As regards the distribution of particular symbionts amongst different stocks andsyngens 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 twomain types, one type being found in a number of stocks of syngen 2, another typein syngen 4, notwithstanding the world-wide geographical range of each of these twosyngens. Sonneborn (1959) has pointed out that, amongst the syngen 4 killer stockscontaining 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, exhibitsrelatively few examples of symbionts, but out of the five in our present collectionsfour are mus, though each of these is distinct judged both by morphology of theparticles 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, onefrom Florida, the other from Uganda, bear almost identical symbionts of a highlycharacteristic type (gamma). These are so far the only known occurrences of thissymbiont. In view of its potent killing properties, it is unlikely that other exampleswould have been missed, had they been present in laboratory collections.

Finally, the delta particles of stock 561 (syngen 1) and stock 225 (syngen 6) aremorphologically almost indistinguishable in spite of their widely different origins.

Some syngens have not been found to contain symbionts at all. An example issyngen 9, of which many Scottish stocks have been collected from the same ponds andstreams as syngen 2 stocks, which very frequently contain kappa particles.

Thus the distribution of the different types of symbiont amongst the stocks andsyngens of P. aurelia is markedly non-random. The implications of this are atpresent 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 themeans by which they have arrived at their present wide, and in some cases world-widedistribution (Sonneborn, 1957). Individual paramecia must remain in fresh waterat all times or they die. So far as we know, transport from one place to another isaccidental and probably rare. Most populations in enclosed waters are presumed to beisolated. Even less do we know how the symbionts are spread about. One way, ofcourse, 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-


bionts readily lose them irreversibly. Re-infection must be exceedingly rare, since thedensity 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 betweensymbionts and paramecia. For example, syngen 2 and syngen 9 are two commonsyngens in Scotland and often found in the same sample of water. Syngen 2 stocksnearly always contain symbionts; syngen 9 has never been found to contain them.Secondly, there is the characteristic association of certain types of symbiont andcertain syngens, as mentioned above. Thirdly, there are many examples of symbiontswhose maintenance depends on the presence of a specific Paramecium gene. Thiswas shown first in the case of 51-kappa (Sonneborn, 1943), and subsequently forvarious 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 ofparamecia contain symbionts, as already mentioned. This raises the question of thevalue of the symbiosis to either member of the partnership. The symbionts obviouslyget a convenient and abundant supply of nutrients, and if capable of resisting digestionby 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 indeeda striking fact that enormous numbers of 'foreign' micro-organisms, occupying anappreciable 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 somesymbionts aid their hosts by synthesizing some nutrient which the paramecia wouldotherwise have to derive from the medium, and this could be so in the case of lambdawhich, 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 significancein nature, owing to the low densities of ciliates normally found. Killers and sensitivesare known to co-exist in the same small sample of water. On the other hand, parameciawhich contain symbionts are immune to the killing action of symbionts of the sametype, when present free in the water. Thus such paramecia would to some extent beprotected, though presumably against only a minute proportion of the possible toxicmicro-organisms in the environment.

Whatever their ecological significance, these symbiotic associations are very commonin 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 andOxytricha bifara (van Wagtendonk & Soldo, 1965). Kirby (1941) describes a numberof examples of other protozoan symbionts. Since ciliates are continuously imbibingbacteria-laden water, it might be thought that the environment of these organisms isparticularly favourable for the establishment of these symbioses. However, similarphenomena may be quite common in other groups of animals. Buchner (1965) claims


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 insectsymbionts (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 ofsome strains. Finally, Woods & Bevan (1968) have described a killer factor in yeast.

If such occurrences are widespread, and if the symbionts have toxic effects, theimportance 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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BEALE, G. H. & JURAND, A. (i960). Structure of the mate-killer (mu) particles in Parameciumaurelia, stock 540. J. gen. Microbiol 23, 243-252.

BEALE, G. H. & JURAND, A. (1966). Three different types of mate-killer (mu) particle inParamecium 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 relationto 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, variety4. Genetics, Princeton 39, 229-239.

HECKMANN, K., PREER, J. R. & STRAETLING, W. H. (1967). Cytoplasmic particles in the killersof 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: ColumbiaUniversity Press.

KUNG, C. (1968). Oxidative Metabolism of Kappa Particles from Paramecium aurelia, Stock 51in 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 insecteshomopteres. C. r. liebd. Se'anc. Acad. Sci., Paris 264, 695—69S.


MUELLER, J. A. (1962). Induced physiological and morphological changes in the B particle andR body from killer paramecia. J. Protozool. 9, 26.

MUELLER, J. A. (1963). Separation of kappa particles with infective activity from those withkilling activity and identification of the infective particles in Paramecium anrelia. Expl CellRes. 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 Parameciums; 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.

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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.

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(Received 11 October 1968)

ABBREVIATIONS ON PLATES

al, alpha particle mi, micronucleusB, bright kappa particle mu, mu particlescaps, ' capsomeres' N, non-bright kappa particlescyt, cytoplasm R, refractile body in bright particlefl, flagella s, ' sheath' to R bodyfv, food vacuole containing bacteria sb, ' small bodies' in macronucleusIb, ' large bodies (' nucleoli') in macronucleus v, virus-like particles in R bodiesma, 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. Mutantshowing only N particles, x 3000.

Fig. 10. 51 -kappa (syngen 4). Electron micrograph showing transverse section throughR body, x 60000.

Fig. 11. 51-kappa (syngen 4). Electron micrograph showing longitudinal section throughR body, x 60000.


10*

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

12Fig. 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.

83Endosymbionts of Paramecium

20Fijj;. 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). f l u s t e r s 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 andflagella 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 andflagella 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 membranetrailing off into cytoplasm (arrow), x 60000.


G. H. Beak, A. Jurand and J. R. Preer

Fig. 32. 225-delta (syngen 6). Osmium-lacto-orcein. Dark phase-contrast. Long andshort 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 materialon the outer membrane (arrow), x 57000.

Fig- 35- 561-delta (syngen 1). Electron micrograph, x 56000.


\ ^

* *>i!

its'


Fig. 36. 562-alpha (syngen 2). Sickle-shaped particles in macronucleus of growingparamecium. 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 slenderform, x 60000.


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THE CLASSES OF ENDOSYMBIONT OF PARAMECIUM AURELIA