The Classes of Endosymbiont of Paramecium Aurelia
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J. Cell Sci. 5, 65-91 (1969) 65 Printed in Great Britain THE CLASSES OF ENDOSYMBIONT OF PARAMECIUM AURELIA G. H. BEALE AND A. JURAND Institute of Animal Genetics, Edinburgh 9, Scotland AND J. R. PREER Department of Zoology, Indiana University, Bloomington, Indiana 47401, U.S.A. SUMMARY The endosymbionts of Paramecium aurelia appear to consist of a number of different Gram- negative bacteria which have come to live within many strains of paramecia. It is not known whether in nature this relationship is mutually beneficial or not. The symbionts from one paramecium may kill other paramecia lacking that kind of symbiont. We identify the following classes of endosymbiotic organisms. First, kappa particles (found in P. aurelia, syngens 2 and 4) ordinarily contain highly characteristic refractile, or R, bodies, which are associated with the production of a toxin which kills sensitive paramecia. In certain mutants of kappa found in the laboratory both R bodies and ability to kill have been lost. Second, mu particles (in syngens i, 2 and 8) produce the phenomenon of mate-killing. Third, lambda (syngens 4 and 8) and sigma particles (syngen 2) are very large, flagellated organisms which kill only paramecia of syngens 3, 5 and 9, and are enclosed in membrane-bound vacuoles. Fourth, gamma particles (syngen 8) are minute endosymbionts, surrounded by an additional membrane resembling endoplasmic reticulum. They have strong killing activity but no R bodies. Fifth, delta particles (syngens 1 and 6) possess a dense layer covering the outer membrane. At least one of the two known stocks is a killer. Sixth, nu particles are a heterogeneous group of particles (syngens 2 and 5) which do not kill or possess distinctive morphological characteristics. Seventh, alpha particles (syngen 2) are the only known nuclear symbionts of P. aurelia; they are found in the macronucleus. Alpha is also exceptional in being the only particle which is highly infectious, though certain of the other symbionts can also be taken up by paramecia lacking them, under special conditions. INTRODUCTION The object of this paper is to survey the different kinds of endosymbiont which grow in the cytoplasm or macronucleus of Paramecium aurelia. In using the word 'endosymbiont', we simply mean one organism living within the cells of another, and do not imply anything about the interactions between host and symbiont, whether beneficial or harmful. Knowledge of the symbionts of P. aurelia stems from the dis- covery by Sonneborn (1938a), that certain strains of the ciliate are 'killers'. The killer paramecia were originally inferred to contain kappa particles in the cytoplasm from the inheritance of the killer trait (Sonneborn, 1945). It was shown that, due to the presence of kappa, a toxic material, at first called 'paramecin', was released into the water, and sensitive paramecia in the vicinity were damaged or killed. Later, kappa particles were identified under the light microscope by examination of fixed and stained paramecia (Preer, 1950) and by observations with the phase-contrast microscope of unstained crushed paramecia (Preer & Stark, 1953). 5 Cell Sci. 5 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'.