J. Cell Sci. I, 31-34 (1966) 3 IPrinted in Great Britain

THREE DIFFERENT TYPES OF

MATE-KILLER (MU) PARTICLE IN

PARAMECIUM AURELIA (SYNGEN 1)

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

SUMMARY

A new rapid method for staining endosymbiotic particles in Paramecium is described. Twonew strains of mu particle in P. aurelia syngen i are compared with the previously known muparticles of stock 540 by light and electron microscopy. Morphologically the three types ofparticle differ only in size, but each converts its host Paramecium into a mate-killer of a specifictype. Each of the three particles appears to require the presence of different genes of Paramecium.

INTRODUCTION

The bacterium-like endosymbionts called mu particles, which grow in the cytoplasmof Paramecium, and cause the latter to become a 'mate-killer', were first described bySiegel (1953), working with P. aurelia, syngen 8. Later a similar type of particle wasfound ih stock 540 of syngen 1 (Beale, 1957), and some details of these mu particlesas observed by light and electron microscopy have been described (Beale & Jurand,1960). Current interest in these particles centres around two problems: (1) the homo-logies of the particles with other micro-organisms; and (2) the dependence of theparticles on the genetic apparatus of the host paramecia. For the further study of theseproblems it is desirable to obtain a number of different types of particle in stocks ofthe same syngen of P. aurelia, capable of being intercrossed for genetic analysis.Making use of a new, rapid method for staining the particles, whereby a large numberof stocks can be screened in a short time, all the old stocks of syngen i, as well as somenewly collected material, have been examined. This search has resulted in the dis-covery of a number of new endosymbionts, some of which are now described.

MATERIALS AND METHODS

Three stocks of P. aurelia, all mate-killers, were studied: stock 540, collected inMexico in 1956 (kindly supplied by Dr T. M. Sonneborn); stock 548, collected atLake Malibu near Los Angeles in 1962; and stock 551, collected in Golden Gate Park,San Francisco, in 1964. In addition, sensitive stock 513, lacking cytoplasmic endo-symbionts, was used for testing the mate-killing effects of the other stocks.

The paramecia were grown in the standard bacterized lettuce medium (Sonneborn,

Staining of mu particles was carried out as follows. The paramecia were placed in a

32 G.H. Beak and A. jfurand

small drop on a slide, as much of the fluid as possible was withdrawn by a micro-pipette, the paramecia were lightly fixed by exposure to OsO4 vapour for a fewseconds, and immediately stained with a small drop of full-strength lacto-orcein (i gsynthetic orcein, 22 ml lactic acid, 36 ml glacial acetic acid, 42 ml distilled water).A coverslip, with vaseline smeared around the edges, was placed over the drop ofstained paramecia and lightly pressed down, flattening but not disrupting the animals.The preparations were examined under phase contrast with a x 100 oil-immersionobjective. This method has been found to show clearly all types of endosymbionts(mu, kappa, etc.) as well as the micronuclei, which are ordinarily difficult or impossibleto identify in temporary preparations. The method has the advantage of making aclear distinction between symbiotic particles in the cytoplasm of the paramecia, andbacteria in the food vacuoles.

For electron microscopy, the paramecia were fixed in 1 % osmium tetroxide (withacetate-veronal buffer at pH 7-2, and 10 mg/ml CaCl2 for better preservation ofmembranes) and embedded in Araldite. After sectioning, the material was stained with1 % aqueous KMnO4 solution containing uranyl acetate.

RESULTS AND DISCUSSION

The three types of mu particle—540, 548 and 551 mu—are illustrated in Figs. 1-3(light microscope) and Figs. 4-6 (electron micrographs). Particles of type 540 muconsist of rod-like structures, approximately 0-3 fi wide and varying greatly inlength, from about 2-20/1 or more. Starvation of the paramecia results in a greatincrease in the total numbers of mu particles per cell, and causes production of thevery long forms. In actively growing paramecia, the number of particles is smaller,long forms are absent and the particles are usually clumped together (as found also bySiegel (1953) for the mu particles of syngen 8). The two other types, 548 mu and551 mu, are much shorter (1—1-5/* an(^ I ' 5~ 2 /* l°ng> respectively) than 540 muparticles, and neither 548 mu nor 551 mu develop the long forms of 540 mu. The type548 mu forms clumps but 551 mu does not.

Electron microscopy shows that all the particles have a conspicuous outer mem-brane. The internal contents are rather undifferentiated. From previous work (Beale& Jurand, i960) it has been shown that 540 mu particles contain DNA (as well asRNA), spread throughout the particles; and the better electron-microscope techniquesnow available show the presence of extremely fine filaments throughout the particles.Certainly nothing like the differentiated nuclear structures of certain bacteria can beseen here. No obvious differences in the contents or outer membranes of the threetypes of particle can be seen.

Nevertheless each of the particles has distinct properties. Each converts its hostparamecium into a mate-killer, as shown by the fact that conjugation between any ofthe three and the sensitive stock 513 results in death of the ex-conjugant derivingcytoplasm from the 513 parent. Of the three mate-killers, stock 548 has the mostpronounced effect; even at the moment of separation of the ex-conjugants, the sensi-tive one can be seen to be already much reduced in size, and death occurs within a few

Mate-killer particles in Paramecium 33

hours. Stock 540 mate-killer usually causes death of the sensitive ex-conjugant onlyafter about 12 h, and sometimes a fission of the 'doomed' cell may take place. Stock551 mate-killer has a still more delayed effect, causing death only after 1-2 days, andoccasionally the clones from the sensitive ex-conjugant recover completely. This varia-tion in intensity of mate-killing effect recalls some earlier findings by Levine (1953)with three syngen 8 mate-killers, which could be arranged in a ' peck-order' of matekilling. However, the interrelations of the three types of syngen 1 mate-killers de-scribed here differ in that conjugation between any two of them (548 x 551, 540 x 548,540 x 551) results in death of both ex-conjugants (except that occasionally 551 failsto kill the others). This mutual killing effect has a parallel in the effects of varioussymbiotic particles (kappa) studied by Sonneborn (1939) and Preer (1948) in syngen 2.Here killing does not occur as a consequence of mating, but is due to the excretion ofa lethal agent from the kappa-bearing Paramecium into the culture fluid, and subse-quent action of this liberated killing agent on a sensitive animal. It was found that thekillers of stock 7 were sensitive to killing by stock 8, and vice versa. Thus, in this caseat least one type of particle does not confer on a Paramecium immunity to killing byanother type, and the same is true of the three types of mu particle now described.

The genetic basis of ability to maintain mu particles in stocks 540, 548 and 551 is atpresent under study. It has previously been reported (Gibson & Beale, 1961) thatstock 540 contains two dominant genes (Mx and M2), either of which is capable ofmaintaining the mu particles. When these genes were replaced by the two recessivealleles mx and w2 from stock 513, all mu particles were destroyed. Crosses have nowbeen made between the two other mate-killer stocks (548 and 551) and the sensitivestock 513. In each case mu particles were maintained in Fx and segregation of animalswith and without mu particles occurred in F2. Hence it is likely that both 548 and 551contain one or more dominant genes necessary for maintenance of these mu particles.Further, cultures of stock 540 lacking mu particles (and therefore not mate-killers)have been obtained by various environmental treatments (extremes of temperature,penicillin treatment, etc.), and crossed with stocks 548 and 551 containing mu particles.In F2 segregation of animals with and without particles was again found. Hence, thegenes in stocks 548 and 551 supporting growth of their respective mu particles aredifferent from the genes Mx and M2 which support the 540 mu particles. These resultsindicate that there is a high degree of specificity in the interrelations of the muparticles and the genotype of the host cell.

It is now becoming clear that quite a high proportion of wild strains of P. aureliacontain endosymbiotic particles. By making random collections and staining theparamecia by the method described here, many new particle-containing strains havebeen found (which may or may not be killers or mate-killers). Not uncommonly it hasbeen found that after cultivating the paramecia in bacterized lettuce or grass mediumfor a short period in the laboratory (even with slow growth rates of the paramecia),the symbionts rapidly disappear, due presumably to the specific nutritional require-ments of some symbionts.

These facts—the abundance of different types of endosymbionts in natural popula-tions of P. aurelia, the remarkable adaptation between the particles and the host

Cell Sci. 1

34 G. H. Beale and A. Jurand

genotype, and the ease with which some particles are lost from the paramecia—makeit seem likely that in nature there is potentially a vast array of micro-organisms(presumably free-living bacteria) capable of establishing themselves in Paramecium,and that infection and loss may occur repeatedly. Occasionally permanent symbioticsystems are established, and these are the systems available for study.

REFERENCESBEALE, 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. (1960). Structure of the mate-killer (mu) particles in Paramecium

aurelia, stock 540. J. gen. Microbiol. 23, 243-52.GIBSON, I. & BEALE, G. H. (1961). Genie basis of the mate-killer trait in Paramecium aurelia,

stock 540. Genet. Res. 2, 82-91.LEVINE, M. (1953). The diverse mate-killers of Paramecium aurelia variety 8: their interrelations

and genetic basis. Genetics, Princeton 38, 561-78.PREEK, J. R. (1948). Microscopic bodies in the cytoplasm of 'killer' Paramecium aurelia.

Genetics, Princeton 33, 349-404.SIEGEL, R. W. (1953). A genetic analysis of the mate-killer trait in Paramecium aurelia, variety 8.

Genetics, Princeton 38, 550-60.SONNEBORN, T. M. (1939). Paramecium aurelia: mating types and groups; lethal interractions:

determination and inheritance. Am. Nat. 73, 390-413.SONNEBORN, T. M. (1950). Methods in the general biology and genetics of Paramecium aurelia.

J. exp. Zool. 113, 87-143.

{Received 8 October 1965)

Journal of Cell Science, Vol. i, No. i

Figs. 1-3. Three types of mu particle (540, 548, 551 respectively) as seen by phase-contrast microscopy of preparations stained by method described in text. The particlesare seen scattered throughout the cytoplasm, cyt, cytoplasm; ma, macronucleus;mi, micronucleus. x 800.

G. H. BEALE AND A. JURAND (Facing p. 34)

Journal of Cell Science, Vol. i, No. i

540'•'•^.3/

r.

4 >

551

Figs. 4-6. Electron micrographs of the three types of mu particle (540, 548 and551 respectively), surrounded by cytoplasm of Paramecium.

G. H. BEALE AND A. JURAND