Enriques studied the same general problem and reached the conclusion that the degeneration and death of infusorian cultures was due to bacterial poisons, because he succeeded in breeding Glaucoma scintillans for 683 generations without signs of degeneration when he took measures to eliminate this factor. Whether this was the crucial factor in his method is open to question, but the significant fact remains that his animals survived nearly twice as long as those of earlier workers, without conjugation or artificial stimulation, thus suggesting that, if suitable conditions are supplied, reproduction by division can proceed indefinitely.

At this point I took up the problem and first investigated the possibility that the degeneration observed in the previous investigations was induced by too great uniformity in the conditions of culture, or by the culture-medium being deficient in something essential for the continued well-being of the organisms. A race of Paramæcium aurelia was isolated in 1907 and bred on infusions of various materials found in the natural environment of the animal, while a sub-culture was subjected to the relatively constant hay-infusion culture-conditions generally employed. The result was that the cells bred in the constant hay-infusion medium died out after a typical Calkins cycle, while those bred on the 'varied-environment' medium did not pass through periods of marked physiological depression or show morphological changes which could be interpreted as abnormal. This race is still, after more than eight years in culture, in a normal condition, having attained over 5250 generations without conjugation or the use of artificial stimuli.

The success with the varied culture medium naturally led to the question whether the longevity of Paramecium, on a varied environment, is dependent upon intrinsic stimuli from the frequent changes of the medium, or whether a constant medium of hay-infusion is unfavorable because it lacks some elements which are essential for the continued existence of the organism. Accordingly,1 a sub-culture of this race was bred for a period of nine months on a constant culture-medium of beef-extract. The continued health of the organisms on this constant medium throughout the experiment, which was continued sufficiently long to include a Calkins cycle, if such 1 Woodruff and Baitsell: Journ, of Exp. Zool., 1911.

was inherent, indicated that it is the composition of the medium, rather than the changes in the medium, which is conducive to the unlimited development of this race without the necessity of conjugation or artificial stimulation.

From a study of various species of hypotrichous infusoria, as well as the main culture of Paramecium aurelia, it was found that minor periodic rises and falls of the division-rate occur, from which recovery is autonomous. These fluctuations were termed ' rhythms' and contrasted with the so-called cycle, which comprises a varying number of rhythms and, according to Maupas and Calkins, ends in the death of the race, if conjugation or artificial stimulation is not resorted to.

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The problem of rhythms was then studied intensively. It was found that the subjection of the culture to the most constant environmental conditions failed to eliminate the rhythms and thus to resolve the graph of the multiplication rate into an approximately straight line; but, instead, the rhythms appeared slightly more pronounced. It was also found, from a study of the temperature coefficient of the rate of reproduction of the culture, that this is influenced by temperature at a velocity similar to that for a chemical reaction, except when the rhythms interfere. Thus, it is apparent that there are inherent rhythmical changes in the phenomena of the cell which produce slight fluctuations in the division-rate.

The results, then, from the study of this pedigreed race of Paramecium aurelia led us to conclude that this organism, when subjected to suitable culture conditions, has the power of unlimited reproduction by division without conjugation or artificial stimulation; the only necessary variation in the rate of reproduction being the normal minor periodic rise and fall of the division-rate, due to some unknown factor in cell phenomena, from which recovery is autonomous (rhythm).

Calkins, however, did not share this optimism and sought the explanation, of the diametrically opposite results derived from his and from our cultures of Paramacium, in variations in the tendency to conjugate, which he and Jennings had found to exist in different races of this organism. Thus, Calkins emphasized the fact that he could readily induce conjugation in his culture, whereas experiments to secure conjugation in our cultures were without effect. He, therefore, stated that "the two races cannot be compared in regard to vitality, since normal conjugation was prevented in the conjugating race, whereas in the non-conjugating race there has been no artificial prevention of a normal process."

With this issue raised, it was essential to determine whether our race was actually non-conjugating. Accordingly, a more extensive series of mass cultures were started from it, with the result that conjugants were finally secured, thus demonstrating that this race is a conjugating race when the proper conditions for conjugation are realized. Therefore, there is no evidence extant that a non-conjugating race of Paramecium exists.

In a recent paper, Calkins states that possibly his terms "conjugating" and "non-conjugating" were not happily chosen, and that he merely meant to indicate that some races are more prone to conjugate than others. Admitting this interpretation of his terms, they express a fact. But this interpretation begs the question which his suggestion was advanced to explain.

With this theory eliminated, the results derived from this culture demonstrate, we believe, that the very limited periods in which Maupas, Calkins, and others observed degeneration, have no significance for the question as to whether degeneration and death are inevitable results of reproduction without conjugation. In other words, this one positive result from this race outweighs all the negative evidence derived from work on the infusoria, and justifies the statement that these organisms can live indefinitely, when subjected to favorable environmental conditions, without conjugation or artificial stimulation.

With conjugation eliminated as a necessary factor in the life history, obviously the next point to be elucidated, if possible, was the underlying factor inherent in the cell, the physiological expression of which is the rhythm. Although morphological or physiological variations that could be interpreted as the result of degeneration 5 Calkins: Am. Naturalist, 1915.

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were never observed in this race of Paramacium, we early noted "that various nuclear changes which are not at present recognized occur normally in the life history of Paramecium"; and we suggested that possibly, when conjugation is prevented, a reorganization of the nuclear apparatus within the individual cell occurs. Erdmann independently reached an essentially similar position from a consideration of the published data on this culture and a critical study of infusorian life histories; and further, in an experimental study of Amaba diploidea, suggested that a relation exists between sexual phenomena and rhythms. Accordingly, we collaborated in a study of the daily cytological changes of this race of Paramacium during a period of six months, and discovered that the rhythms in the division-rate are the physiological expression of internal phenomena which involve the formation of a complete new nuclear apparatus, by a definite sequence of normal morphological changes that simulate conjugation. This nuclear reorganization, which we term endomixis, consists, in essence, of a gradual disintegration and absorption of the macronucleus in the cytoplasm. Simultaneously, a multiplication of the micronuclei is in progress. Certain of the resulting micronuclei degenerate while the remaining one (or two) form the new macronuclear and micronuclear apparatus. This results in the reorganization of the cell without the fusion of two animals.

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An essential morphological difference between endomixis and conjugation is the absence of the third micronuclear division, which, in conjugation, forms the stationary and migratory micronuclei; and, of necessity, the non-formation of a syncaryon. After conjugation the reorganized cell has a new macronuclear and micronuclear apparatus, composed of combined material from the conjugants, while, after endomixis, the reorganized cell has a new macronuclear and micronuclear apparatus composed of material from its own micronuclei. In a word, the essential distinctive features of endomixis are the absence of the third micronuclear division and

Woodruff: Amer. Naturalist, 1908.

Erdmann: Ergeb. d. Anat. u. Ent., 1908; Archiv. f. Protistenk., 1913. & Woodruff and Erdmann: Proc. Soc. Exp. Biol. and Med., 1914; Journ. Exper. Zool., 1914; also Erdmann and Woodruff: Biol. Cent., 1914.

the absence of the introduction of foreign nuclear (and cytoplasmic) material into the cell.

For reasons advanced elsewhere, we hold that endomixis is not parthenogenesis, but it is not necessary at this time to enter into a more or less academic discussion in regard to the exact classification of endomixis among Entwicklungserregung phenomena.

In the light of the discovery of the details of endomixis, by the daily study of pedigreed cells of this race, a survey of the cells, which had been preserved at intervals during the previous seven years of its life, revealed a number of the crucial stages of endomixis, thus showing that the process has been in progress ever since the race has been bred, and is not, as Hertwig suggests, a development during long subjection to culture. That it is not even a peculiarity of this race is evident from the fact that we have found endomixis in four other distinct races selected at random-three from America and one from Germany. Further, Hertwig, in 1889, incidentally noted in a mass culture, in which conjugation had not been observed for a long time, certain animals whose nuclear structure apparently indicated isolated stages of the process which have been elucidated in our cultures. Therefore, it seems well established that endomixis is of general and probably universal occurrence in Paramecium aurelia. It also occurs, with essentially similar features, in all the races of Paramecium caudatum which we have studied. 10

Now, in regard to the significance of endomixis from the standpoint of our subject-rejuvenescence in protozoa: It is clear that the cycle emphasized by Maupas, Calkins and others, is merely a phantom which has continually receded as each successive investigator has approached the problem with improved culture methods, until it has vanished with this eight-year-old culture. What remains then is the rhythm and in the light of endomixis-the underlying cytological phenomenon of which the rhythm is an outward physiological expression-the whole problem takes on a new aspect. The cell automatically reorganizes itself periodically by a process which, in its main features, simulates conjugation, but without a contribution of nuclear material from another cell.