STUDIES O F A PEDIGREE: CULTTJRE O F PARABIECIUAf CRLKTNSI

HOPE SPENCER O s b o i n Zoological Laboratmy, Y a l e Cnwersrt?j

FOUK. FIGURES

Refiiiemcnts in cultural methods have gradually prolonged the time during which ciliates will live without conjugation in tlie laboratory, until it appears to he positively established that unsuitable environment is the factor which previonsly led to the view that death is inevitable when conjugation is prevented1 However, the study of the life-history of Para- mecium aurelia and Paramecium caudatum has shown that the division rate is punctuated by periodic fluctuations, termed rhythms, which uniform conditions fail to remove.2 Mritli the discovery of endomixis at the low point in tlie rhythms in Paramecium aurelia and Paramecium caudatum, the cause of the rhythms seemed established ; 3 but the fact that endo- mixis has never been described during the free life of certain ciliates, in particular hypotrichs, which apparently can live indefinitely without conjugation although they exhibit rhphms seemingly comparable with those of Paramecium, suggests that this nuclear reorganization phenomenon, as

lWoodruff, L. L., The present status of the long-continued culture of Pxra- mecium aurelia at Yale University, Proc. Nat. Acad. of Sciences, vol. 7, 1921; Woodruff, L. L., and Spencer, H., Studies on Spathidium spathula. 11. The significaiice of conjugation, Jour. Exp. Zool., vol. 39, 1924; Woodruff, L. L., and Moore, E. L., On the longevity of Spathidiuni spathula without endomixis or conjugation, Proc. Nat. Acad. of Sciences, vol. 10, 1924.

Woodruff, L. L., and Baitsell, G. A., Rhythms in the reproductive activity of Infusoria, Jour. Exp. Zool., vol. 9, 1911.

%Woodruff, L. L., and Erdmann, R., A normal periodic reorganization process without cell fusion in Paramecium, Jour. Exp. Zool., vol. 17, 1914.

543

544 H O P E Sl’KXCEW

well a s conjugation, may not lie a iiecessity for tlie contiiiued life of the celL4

Paramecium calkiiisi was found to be f avorablc material for the study of this problem, and accordingly experimeiits were inaugurated in ail attempt to answer tlie questioiis : 1) (’air Paramecium calkinsi live indefinitely without coil- jugation or’ rndomixis ? 2 ) Are rhytlims evident without ciitlornixis ?

The author n-ishes to express to Prof. I , . 1,. Woodr’nff her appreciation of his constructive criticism exteiitliiig through the tliiratioii of the work.

EIIS’I’ORT OF T H E CULTURES

The animal used to start culture I of these experimeiits was takeii on Jlay 26, 1921, from the origiiial Paramecium calkiiisi

Fig. 1 Parameciimi calkinsi, culture I: periods 39 to 36, December 4, 1921, to N;ircli 3, 1922, culture kept a t room tei1iper:rture. Gr:ipli sliowing average di\isioii rate of four lines again : i re raged f o r five d a j s .

ciiltiire’ a d bred, as were all the pedigree cultures, oil

stantlard heef 1 solations were, in general, made a t tlie same time tlailg, aiitl the bacterial flora was kept the same, it is helievetl, hy daily cross-infection. Such exteriial factors as dapliglit and barometric pressure need not he consicl~reci, since they have already heen shown to be without significant effect on the colorless I ’arame~ia.~ Culture I was carried at room temperature until July 21, 1922, when it was stoppctl, after being in pedigree culture 420 days arid attaining 392

Woodruff, 1,. L., An experimclital study of the life liistoi y of liypotriclious ruff, L. L., and Speiiwi, H., loc.

(*it.; Moore, E. L., loc rit. ’ Lf’oodruff, I,. L., The s t ructure , life Iristoiq, :inti iiitmgciieric irlatioiisliips uf

€’:i~:i~iieciuin calkinsi, sp. nor., Uiol. Dull., ~ o l . 47, 1921. ‘ Woodruff, L. li., :ind Baitsell, C. A., Tht. repiodurt ion of I’:~~:inieciunn :turclia

in :I ‘constaiit’ cnltiue medium of heef e u t i x v t , .Tour. Eip. Zool., bol. 11, 1911. ’ T\’c~odruff, L. Id., :rnd B:iitsell, G. A . , Jour . Exp. Zohl., vol. 9, 1911, loe. cit.

PEDIGREE CULTIJIW OF P A D A M E C I C N CAT,RINRI 545

generations. Figure 1 shows a small but typical part of the graph of the division rate.

Culture I1 was started on September 13, 1921, as a suh- culture of culture I a t the 171st generation. The animals were kept in a Freas thermostat at a practically constant temperature. Food, pipets f o r the isolation of the intli-

z

1 U

D 0 Fig. 2 A, the average daily temperature again averaged for five days. B,

Paramecium calkinsi, culture 11. D, Pam- niecium aurelia. All cultures kept a t the same temperature and nnder ‘constant’ conditions durinq periods 39 to 36, Decemhcr 4, 1921, t o March 3, 1922. Method of plotting the s:inie as in figure 1.

C, Par2mecium calkinsi, ciilturp 111.

viduals, and slides to wliich they were transferred were also kept in the same thermostat further to minimize changes in temperature. On June 11, 1922, culture IT died at the 420th generation, having lived synclironously with culture I for 272 days (fig. 2, B) .

On September 20, 1921, culture I11 was isolated from stock of the original culture in hay infusion, and was bred on beef extract until it died on Fehrnary 25, 1922, having lived 154

c lay and attained 145 generations. The environmental c o w ~litioiw of this culture wei*e the same as those for culture 11; that is, the animals were kept in the same thermostat aiitl bred 011 the same supply of beef (fig. 2, C).

(’nlture T V was started as n subculture from culture 11 oii April 29, 1922. These animals were bred to determiiit. the itifluetice of different eiivirorimeiital coriditions on tlie flnc tiiatioiis in tlie division rate. Accordingly, tlie culture was carriecl in ariotlier room n-here the temperature was not thc same and a diflf’erent bacterial flora was supplied, although tlie hasic medium, standard beef extract, was used as heforc. Those lilies were stopped after 80 tla;vs, wlicii they liatl rcachctl 54 generations (fig. 3).

c- -

Fig. 3 I’ninniecium mlkiiisi, culture IV (---), periods 63 to 76, March 30, 1!122, to Juiie 11, 1922, using ciiltnre I1 (-) for comparison daring s?n- chronous pvriods. C’onditions (ti\ erst’.

F o r comparison with P. calkinsi cultures, two cultures (V and VI) were started from Woodruff’s original Para- mecium aurelia culture on September 15, 19217 and car.ried hi tlie Freas thermostat until the end of the definitive experi- ments 011 July 21, 1922. Endomixis was readily demonstrated soon after the cultures were under observation, tliereb>* sliov- iiig that the reorganizatioii occurred as usual at tlir low poiiits in tlie rhythms in Paramecium aurelia (fig. 2, D) .

COXJUG ATION

T n an attempt to induce conjugation in this race of P. calkiiisi stock flasks of the animals in hay infusion have I~eeii maintained throughout the entire history of the culture aiid

up to the present time-May, 1924. Material from the top, niitldle, aiid hottom of tlie flasks was examiiied practically daily for a period of about two months. Further observations m-ere made at varying intervals during tlie rest of the more

PEDIGREE CULTURE O F PBIiAMECIUM C A L R I N S I 547

tliaii tliree years over which this study has extended. Not a single pair of coiljugants was seen, and accordingly it may he accepted with practical surety that conjugation did not occur.

Since ZweibaumR stated that eonjugation was most readily eiTected by allowing the animals to starve for some weeks, and then treating them with a salt, notably with N/50000 FeC1, and N/1200 NaC1, the same procedure was followed with 1’. calkirisi. I n some cases these solutions were alone used as media, in others in combination with beef or hay. Both starved and well-fed animals were employed. Not a single pair of conjugants resulted.

T n experimental work on Paramecium, Hopkins9 f ouiid that N/1000 NaNO, aided in bringing about conjugation. Accortl- ingly, animals, both well-fed aiid starved, were subjected to varying strengths of this salt in numerous combinations with hay and beef. These conditions failed to produce conjugants.

Jeiiniiigs“’ stated that old, starved cultures, if revived with fresh food until they are in a heathy condition and then sud- deiilp starved, will frequently provide epidemics of coqjuga- tion. This procedure was carried out in detail and also with certain modifications. Sometimes animals which had been starved in beef were made normal again by a sudden change to hay infusion and rice versa. Rapid starvation was accom- plished either by changing the culture to an old medium, the same kind in wliich it had been living, or to another, or by putting the animals into distilled water. Slow starvation was realized by allowing a heavy culture to exhaust all the avail- able food or by putting the cultures on ice so that the bacterial food supply could not develop so readily. Furthermore, heavy cultures were allowed to evaporate at different rates. All these methods proved of no avail ; conjugation did not occur.

Zweibnum, J., Les conditions nCcess:riies et suffisaiites pour la coiijugason du Arch. f . Protistenk., Rd. 26, 1912.

Hopkins, H. S., The coriditioiis for coiljugation in diverse rnccs of P:ira-

Jour.

Pnramccium caudatum.

mecium.

Eup. Zool., vol. 9, 1910.

Jour . Exp. Zool., 1701. 34, 1921. I” Jeiiiiings, €1. S., Wliat conditions induce conjugation in L’armneciuni.

. 1 O V K S A L OF I \ l ( ~ K I ’ R O L 0 ( s 1 4NI) I’HPSIOIIO(IT. 7 012 ‘39, hO. 2

548 H O P E SPENCER

Siiicc X.’oodruff’s Paramecium aurelia culture was iiiitleu observation for about six years before eonjugation was ohserved,ll it is iiiadvisable definitely to coiiclude that tlie plieriomeiioii cannot occur in this race of P. calkinsi, but it is certain that conjugation can be dispensed with for long periods of time without injury to the iiormal processes of the cell, as indicated by the usual criterion-the division rate. Tlie lack of conjugation is remarkable, since tlie ciliates already studied, e.g., Spathic1ium,l2 which do not exhibit endo- mixis in the active phase of their life-history, usually coii- jiiga te frequently.

ENDOMIXIX

A 1 1 aiialysis of the cytological data accumulated during tlie study of the cultures was made with special reference to endomixis. As briefly reported by Woodruff, about 1000 preserved specimens of P. calkinsi, taken at various points in the life-history, were examiiied f o r evidence of endomixis. Ihriiig a period of about a moiitli, nearly every sister cell of the main line was stained aid studied cytologically. TIIIIS, the possibility of failing to find endomixis, were the plieiiome- 11011 present, was practically precluded, but iio evideiicc of eiidomixis was found.13

In the present series of experiments, over 200 preservccl animals were examined. These were killed chiefly at the low points in the division rate, wliere endomixis in P. aurelia has heen slio\vn to occur regularly, but in no case have there been departures from the iiuclear structnre typical of the vegeta- tive animals. It can therefore be definitely reaffirmed that ciiclomixis docs not occur in P. calkiiisi during active life.

1x1 some forms, notably Didinium iiasiitum, Uroleptus mobilis, aiid Xpathidium ~ p a t l i u l a , ~ ~ eiidomixis has heen found

Woodruff, 1,. L., On so-called conjng:iting :mcl noii-eoiijugnting races of

Woodruff, I,. I,., and Spencer, H., loc. cit. I’aisrrieeinm. Jour. Ehp. Zool., vol. 16, 1914.

lJ Woodruff, L. L., Biol. Bull., loc. cit. l4 C:rlkins, G. N., Didinium nasutum. I. The life histoiy, Jour . Esp. Zobl.,

vol. 1!1, 1915 ; Uroleptus mobilis Engelni. 11. Rmiewnl of ritalit) through C O I I -

jng:rtion, .Jour. Exp. Zool., vo1. 29, 1919; N(JOW, E. L., l o p . cit.

PEDIGREE CULTURE O F PARAMECIUM CBLKIKSI 549

to occur solely during encystment. This raises the question whether P. calkinsi encysts and, if so, whether endomixis occurs during encystment.

A few cases of encystment in Paramecium of different species have been recorded by Prowazek, Simpson, arid others,lj hut these observations have not been corroborated. Indeed, numerous intensive studies on P. aurelia and P. caudatum have failed to show positive examples of encyst- ment, and it may be accepted that encystment docs not occur. Likewise, in the present experiments on P. calkinsi, careful study of animals under many kinds of environmental condi- tions has not revealed a single cyst. Thus, there is no evi- dence of encystment in P. calkinsi, and therefore eiidomixis obviously is precluded from this stage in the life-history. Accordingly, it may be concluded that eiidomixis does not occur a t all iii the life-history of P. calkinsi.

RHYTHMS

-4lthougli eiidomixis is not present in this race of P. cal- kinsi, the graphs of these cultures show that flactuations in the division rate occur. In order to determine whether these fluctuations, in the absence of endomixis, are due to extrinsic or intrinsic factors, a comparison of the several cultures was made.

A study of the graphs of the P. calkiiisi cultures reveals the fact that fluctuations in the rate of reproduction a re present which apparently a re indistinguishable from the rhythms of P. aurelia and P. caudatum. Since the environmental condi- tions of the cultures compared were as near identical as is possible under the conditions of the experiment, there was afforded a favorable opportunity for synchronous rliythms to occur, if these fluctuations are fundamentally extrinsic in origin. From figures 1 and 2 it will be seen that the low points in tlie fluctuations of tlie division rate clo not occur

lo Promazek, R., Kleine Protozoenbeohn chtungen, Zool. Anzeiger, Bd. 22, 1899 ; Simpson, J. Y., Studies on Protozoa; notes on the intimate striicture of Protozoa, as exhibited by intravital staining, Proe. Scot. Mie. Soe., vol. 3 , 1901.

syicliroiiously with aiiy degree of regularity in tlie various cultures being carried simultaneously, and this fact is e q u a l l ~ evident tliroiigliout tlie whole history of the cultures. Tliere is a similar lack of coincidence in the high points in tlw dirision rate. Temperature is the environmental factor to wliicli one mould riatiirally turn f o r tlie cause of the flnctnn- tions in tlie division rate, hut it will be noted tliat in tho graph tlic temperature shows very slight variation for the entire latter half, yet the variations in the division rate of tlie several cultures during this time indicate the usual fluc- tuations. All t l i ~ data, therefore, seem to show that external cotitlitions do not cause rhythms.

DISCCSSION

From a consideration of the foregoing experiments, tlic coticlusion is patent that conjugatioii is not necessary for the coiitinued life of this race of P. calkinsi, as already proved for 1’. aurelia. Of the pedigree cultures, only one, culture I , was alivc at the termiliation of the definitive experiments, hut of tlie others, culture IV had been stopped just previously, atid cultures TI a i d I11 Iiad died. The pedigree culture of P. calkiiisi cai.riecl by Woodrufl had also died. However, at any time during the experimeiits tlie animals were rcadil) cnltnred iii small mass cultures in hay infusion. These h a y infusion cultures, carefully watched to preclude the possihi1it)- of conjugation occurriiig unnoticed, a re still tinder observa- tion arid dividing actively. This fact renders it impossible to interpret tlic death of the slide cultures as a result of degeneration, which is implied in tlie conception of a cycle, but rather iiidicates tliat the medium employed, known to be 1iiglil~- suitable f o r P. aurelia, is, ho.wever, not perfectlj- favorable for P. calkinsi. ilccordingly, no evidence is afforded hy this study of ail inherent ‘cycle’ in the life-his- tory of P. calkinsi which terminates in death if coiijng a t’ 1011

is prevented. Eynallp coriviticing is tlie evidence that eiidomixis does not

occi~r at any period iti tlic life-history of P. calkinsi, a11tl

PEDIGREE CULTUIX OF PARARIECIUM CALliINSI 551

accordingly the coiiclusion is niiavoictable that neither coii- jugation nor endomixis is indispensahle for the iiidefiiiite continualice of reprocluctioii by division in this species of Paramecium.

But altliough eiidomixis does not occur, there are periodic fluctuations in the division rate which are not tlistinguisli- able from those described in various iiifusoria by Wood- rufff’” and defined as a “minor rise and fall in the fission rate t h e to some urikriowii factor in cell metabolism from which recovery is autotomous. ” Later, Woodruff and Erdmann, wlien they discovcred endomixis, believed that this nuclear iwrganization was the uiikiiown factor responsihle for the rhythms. Undoubtedly in P. aurelia and P. caudatum endo- mixis is syiichroiious with the low points of the rhythms, but that endomixis is solely responsible for rhythms is im- probable in view of these results with P. calkiiisi. I n this species, which does not exhibit endomixis, fluctuations occur which would seem to be rhythms, in the sense that term was emploj-etl hy Woodruff in 1905.

SUMMAKY

1. Conjugation has not occurred during four years that Paramecium calkiiisi has been uiitler observation, ant1 all aitempts to induce it have failed.

2. Hridomixis does riot occur in this race of P. calkiiisi aiitl the race can live indefinitely without this process.

3. Eiicystment does not occur in this species. 4. Since neither endomixis nor conjugation has occurred

in P. calkiiisi under laboratory coiiditioiis aiitl the animal s h o ~ v s every indication of the potentiality to reproduce lip division indefinitely, it is clear that neither plieiiomenon is

~iiccessary for the successful contiiiuation of life. ;>. Fluctuations in the division rate, which are regarded

as rhythms, occur, although eiidomixis is absent. Constant coiiditioiis do not eliminate them, aiid therefore rliytlims a r e tlue to some factor iiihereiit iii the organism other than ciidomixis.

18Woodr~ff , L. L., Jour . Esp. Zobl., 190;i, loc. cit.