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Some aspects of conjugation in Stentor coeruleus Webb, Terry Lavern 1968

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SOME ASPECTS OF CONJUGATION IN STENTOR COERULEUS by TERRY LAVERN WEBB. B.Sc, University of B r i t i s h Columbia, 1966 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of ZOOLOGY We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August, 1968 In presenting th is thesis in pa r t ia l fu l f i lment of the requirements for an advanced degree at the Univers i ty of B r i t i sh Columbia, I agree that the Library sha l l make i t f ree ly ava i lab le for reference and Study. I further agree that permission for extensive copying of th is thesis for scholar ly purposes may be granted by the Head of my Department or by h.iJs representat ives. It is understood that copying or publ icat ion of th is thesis for f inanc ia l gain shal l not be allowed without my wri t ten permission. Department of Zoology  The Univers i ty of B r i t i sh Columbia Vancouver 8, Canada Date August; 27, 196$ ABSTRACT i i Conjugation i n Stentor coeruleua was investigated using two experimental methods which regularly yielded large numbers of mating pairs. One method involves a p a r t i c u l a r culture technique, the other requires a mix-ture of c e l l s from dif f e r e n t stocks. Mating pairs ap-peared i n the form of bursts of conjugation, either induced by mixing certain stocks or occurring spontaneously i n some stock cultures. Spontaneous bursts, i n the majority of cases, occurred during a d e f i n i t e i n t e r v a l i n the development of a culture. Morphologically d i s t i n c t pre-conjugator c e l l s appear immediately before as well as during the i n i t i a l stages of a burst of conjugation. Mating pairs were formed by the union of two pre-conjugators. Mixing eight stocks i n a l l possible combinations of twos and observing t h e i r subsequent response revealed they were separable into two complementary mating types. The majority of mating pairs formed i n mixtures of stocks consisted of individuals of di f f e r e n t mating types. Evidence i s presented which i s compatible with the hypo-thesis that c e l l to c e l l contacts between individuals of d i f f e r i n g mating type are necessary for the i n i t i a t i o n of a mating reaction. i i i TABLE OF CONTENTS Page INTRODUCTION 1 METHODS AND MATERIALS 4 1. Stocks of Stentor 4 2. P e t r i Dish Culture Technique 4 3. Photography 6 4. Induction of Conjugation 6 RESULTS 7 1. The Appearance of Conjugation i n Stock Cultures 7 2 . Pre-conjugators and the Formation of Mating Pairs 14 3. Production of Selfing Clones 20 4. Induced Conjugation 21 5. Specific Mating Between Stocks 24 6. Demonstration of Cross-mating 26 7. Role of C e l l to C e l l Contacts i n the Induction of Conjugation 31 8. Survival of Exconjugants 36 9. Abnormal Mating 38 DISCUSSION 40 SUMMARY 51 BIBLIOGRAPHY 52 APPENDIX 56 iv LIST OF TABLES Table Page I The appearance of mating pairs i n stock cultures. 9 II Number of days from the i n i t i a t i o n of cultures to the f i r s t appearance of mating pairs i n stocks CH, H, S, T, and W 13 III Intraclonal conjugation (selfing) i n cultures started from single c e l l s . 22 IV Specific mating i n mixtures of stocks. 25 V Observed classes of mating pairs scored on the basis of r e l a t i v e pigmentation, presence of carmine, and r e l a t i v e size. 30 LIST OF FIGURES Figure Page 1. Estimated change i n the percentage of the population involved i n con-jugation through a spontaneous conjugation burst. 11 2. Representatives of normal vegetative individuals and pre-conjugators. a) Ventral view of a normal c e l l . 15 b) Anterior view of a normal c e l l . 15 c) Ventral view of a pre-conjugator. 15 d) Anterior view of a pre-conjugator. 15 3. Anterior view of a pre-conjugator showing the bulge within the folded-down portion of the f r o n t a l f i e l d . 17 4. Two pre-conjugators with the folded-down portion of their f r o n t a l f i e l d s i n close apposition; bulges not yet joined. 17 5. Two pre-conjugators with bulges just joined. 18 6. Contracted c e l l s just before they p u l l apart. 18 7. A sequence showing the development of a mating pair from i n i t i a l adhesion t i l l just before separation, a t o t a l of 28 hours. a) Bulges just united. b) 4 hours. c) 9 hours. d) 21 hours. e) 28 hours, separation came during the following hour. Mating pair from a mixture of stocks C and S. Squashed pair from a mixture of labelled stock S c e l l s and unlabelled stock C c e l l s . Diffusion chamber (a) before wrapping with bolting cloth and (b) after wrapping i s complete. P e t r i dish culture with d i f f u s i o n chambers i n place. An example of a mating t r i p l e t found i n a mixture of stocks C and S. Mating pair with one partner at about stage 7 of d i v i s i o n . ACKNOWLEDGEMENTS v i i I would l i k e to thank my supervisor Dr. D. W. Francis for his patient guidance and encouragement during this study. I am indebted to Mr. Charles M. Harden f o r allowing me to read his paper while i t was i n manuscript form. I am grateful to Dr. Vance Tartar and Dr. T. M. Sonneborn for t h e i r useful comments and c r i t i c i s m during the study. Appreciation i s expressed to Dr. A. B. Acton, Dr. C. V. Finnegan, and Dr. D. Suzuki for th e i r c r i t i c a l reading of the manuscript. INTRODUCTION 1 An essential characteristic of a d i p l o i d organism i s i t s a b i l i t y to effect, i n one way or another, the recombin-ation of i t s genes i n i t s offspring. Among the c i l i a t e d protozoans t h i s i s brought about by the sexual process of conjugation which involves the temporary union of two indiv-iduals, during which they exchange parts of the nuclear apparatus. The phenomenon of conjugation has been observed and studied i n a large number of c i l i a t e d protozoans. However, information concerning sexual reproduction i n Stentor i s lacking... Conjugation i n Stentor coeruleus has been observed by Moxon (1869), Balbiani (1891), Hamburger (1908), Mulsow (1913), and more recently by Tartar (1961), B u r c h i l l (1967), and Harden and Holland (1968). The early workers found that exconjugants did not survive long after engaging i n sexual reproduction. Mulsow,, however, was able to keep them al i v e long enough to examine the cytological events of conjugation. His studies showed that the events of conjugation accomplish the recombination of chromosomes i n a manner similar to that found i n other ciliates;. namely, the fusion of haploid pro-nuclei and subsequent renewal of the macronucleus from micro-nuclear derivatives. Tartar i n reviewing the subject suggests that the proper conditions f o r conjugation seldom occur. He draws attention to studies i n which Stentor cultures have 2 laeen carried on continuously f o r several years with very few or no mating pairs detected. Recently, two separate studies ( B u r c h i l l , 1967; Harden and Holland, 1968) reported the occurrence of abundant con-jugation i n mass cultures of Stentor coeruleus. B u r c h i l l found that conjugation occurred most often i n cultures which had been fed recently, observing up to f i f t y percent of population involved i n conjugation at one time. Harden and Holland (1968) discuss the effects of conjugation on a large number of conjugating pairs, stating that S. coeruleus seems to be able to follow one episode of conjugation with another a f t e r a short i n t e r v a l . They also observed a high mortality rate among exconjugants but were unable to demon-strate any increase i n mortality rate due to inbreeding. To date, a l l studies of sexual processes i n Stentor have been confined to the occurrence of conjugation i n mass collections or cultures. Such studies, although providing a considerable amount of knowledge, are limited because the occurrence of conjugation i s rather unpredictable. Recent attempts to induce conjugation (Tartar, 1961; B u r c h i l l , 1967) i n t h i s genus have been unsuccessful. In several c i l i a t e genera conjugation has been brought under stringent experimental control by vir t u e of the fact that mating type d i f f e r e n t i a t i o n s account f o r the union of c e l l s . When cultures of complementary mating type are mixed a mating reaction occurs because of cross-mating between 3 the two types. U n t i l the pa r t i c u l a r mating system of a species i s known the controlled matings required f o r genetic studies are impractical. The purpose of th i s study was to investigate sexual reproduction i n Stentor coeruleus with the aim of develop-ing an experimental system with which to control conjugation, and to add to the general knowledge of conjugation i n this genus. The .study was occasioned by the unexpected discovery of persistent conjugation i n stock cultures of th i s c i l i a t e . 4. METHODS AND MATERIALS 1. Stocks of Stentor The study was conducted using nine di f f e r e n t stocks of Stentor coeruleus which were obtained from various sources (see Appendix). They were designated as follows: C, DF, S, SC, ST, T, CH, H and W. To provide genetic uniformity, each stock was established i n culture from the vegetative progeny of a single c e l l (thus representing a clone) and thereafter v/as maintained separately i n continuous culture. Five of the stocks ( C , DF, S, SC, T) were genetically d i s t i n c t because each one was derived from a d i f f e r e n t natural pop-ulati o n . The c o l l e c t i o n s i t e s range from the west coast to the east coast of North America; therefore these stocks should represent a wide range of genetic d i v e r s i t y . Stocks CH and H were o r i g i n a l l y derived from stock T and had been maintained i n separate laboratories for a period of at least two years before being established i n culture for the present study. Stock ST was o r i g i n a l l y derived from organisms belong-ing to stock SC but was also maintained i n a separate labor-atory before being established i n culture for the present study. The o r i g i n of stock W i s not known. 2. P e t r i Dish Culture Technique After being established i n culture each stock was main-tained i n continuous culture by the following subculture 5 method. A l l cultures were maintained i n s t e r i l e p l a s t i c p e t r i dishes (100 x 25 mm). Each p e t r i dish was coated on the bottom with a thin layer of one per cent agar (De Terra, 1966). The agar layer appeared to f a c i l i t a t e the attachment of stentors to the bottom of the p e t r i dish; i n i t s presence consistently good cultures were produced. The culture dishes were prepared by addition of approximately 75 ml of millipore ' f i l t e r e d pond water along with two boiled wheat grains. To i n i t i a t e a culture an inoculum of 0.5-1.0 ml of water, con-taining a species of colorless f l a g e l l a t e s (cultured separate-ly i n the same medium), was added to the culture dish followed 24 hours l a t e r by the introduction of 25-50 stentors. The colorless f l a g e l l a t e s , closely resembling Rhabdomonas costata, acted as food organisms. A l l cultures were maintained i n the laboratory at room temperature .(21°-25°C). Stocks cultured i n this manner consistently yielded a vigorous pop-ula t i o n of rapidly dividing stentors which approached maxi-mum density i n 12-16 days. After reaching maximum density the population entered a stationary phase of growth during which few, i f any, dividing stentors were present. Sub-culturing was carried out when the stentors were well into the stationary growth phase but before the culture began to decline (the culture was then 20-25 days old). A l l cultures were maintained for 30. days regardless of t h e i r time of sub-culture. Cultures were observed daily or every second day 6 to follow their progress from the time of i n i t i a t i o n to the 30 day i n culture. Observations were made by trans-mitted l i g h t using a Wild M-5 stereomicroscope with sub-stage illumination. 3. Photography Organisms were photographed, as they were, i n the culture dishes i n order to record the events of conjugation i n an undisturbed state. Photomicrographs were taken with Kodak high contrast copy f i l m using a Wild Mka 1 camera which was mounted d i r e c t l y on a Wild Mo stereomicroscope. The l i g h t source for photography was provided by a Leitz micro-flesh device with a f l a s h duration of 1/1000 second.. 4. Induction of Conjugation A method was developed which successfully induced con-jugation i n Stentor. The essential feature which i s neces-sary for the induction of conjugation i s the mixing of sex-ually reactive c e l l s from two different stocks. This feature was incorporated into several experiments designed to demon-strate the extent of mating between a l l stocks, the extent of cross-mating between c e l l s of two.stocks, and the actual stimulus which leads to the formation of mating pairs. Dis-cussion of each of these experiments w i l l be reserved f o r l a t e r sections to avoid any confusion which might arise from an awkward separation of a discussion of the methods used and the results obtained. RESULTS 7 The i n i t i a l observations i n t h i s investigation were made with the unexpected discovery of conjugating pairs i n several cultures of stock S stentors. At that time i t was considered possible that the culture technique was providing conditions which favored conjugation. With th i s i n mind a number of subcultures were made using exactly the same technique as before; again, numerous conjugating pairs appeared. These preliminary observations lead to the start of an i n v e s t i -gation which ran from November 1966. to May 1968, the results of which are presented i n this thesis. 1. The Appearance of Conjugation i n Stock Cultures Conjugation, often involving large numbers of c e l l s , was observed frequently throughout the period of study i n stock cultures maintained i n the manner previously described. Cultures of each of nine stocks maintained i n continuous subculture were observed daily or every second day to follow "t h. their progress from the time of i n i t i a t i o n to the 30 day i n culture. After 30 days the cultures were declining and date were no longer recorded. A t o t a l of 602 stock cultures were made and observed; of these, 292 (49%) contained mating pairs at some time during their development. The number of pairs present at one time i n a culture dish varied from less than 1% to greater than 30% of the t o t a l population. These figures are derived from 8 the accumulated data for a l l nine stocks and, as such, are somewhat misleading. They are included here merely to indicate that the culture method does appear to provide conditions which are favorable for sexual reproduction. A more detailed inspection of the data obtained from the consideration of each stock i n d i v i d u a l l y , revealed a de f i n -i t e v a r i a t i o n between stocks i n their'tendency to produce cultures which contained mating pairs (Table I ) . These date show a well, defined tendency f o r some stocks (CH, DF, H, S, T and W) to produce subcultures which regularly contain mating pairs, but at the same time show other stocks (C, SC and ST) to have l i t t l e or no tendency to produce cultures which contain mating pairs. On t h i s basis, the stocks can be divided into two groups: those which regularly conjugate within stock cultures, and those which do not. This d i v i s i o n i s further substantiated by the observation that conjugation i n cultures of stocks C, SC and ST never involved the forma-tion of more than f i v e mating pairs i n any culture, while, i n contrast, conjugation i n cultures of stocks CH, DF, H, S, T and W usually involved the formation of large numbers of pairs, often numbering i n the hundreds. Pe t r i dish cultures i n i t i a t e d with food organisms and stentors usually exhibited a d e f i n i t e sequence of events with respect to their growth pattern and the appearance of mating pairs through the 30 day observational period. During the f i r s t few days the food organisms multiplied rapidly followed closely by an increase i n the number of 9 Table I . The appearance o f mating p a i r s i n s t o c k c u l t u r e s . t o t a l number o f number o f s u b c u l t u r e s s t o c k s s u b c u l t u r e s made c o n t a i n i n g mating p a i r s C 51 6(12%) CH 40 19(48%) DF 11 8(73%) H 123 85(64%) S 242 104(43%) SC 19 1(5%) ST 19 0 T 47 44(94%) W 50 25 (,50%) 10 stentors. The population of stentors approached maximum density Ces t ima ted from the decreasing number of dividing individuals) after about 14-16 days. During the following f i v e or six days the population entered a stationary phase during which few dividing stentors appeared. As mentioned previously, by the time a culture was 30 days old i t usually had begun to decline. When mating pairs appeared i n a c u l -ture dish, they most frequently did so i n the form of a burst of conjugation l a s t i n g several days. In any conjugation burst a large number of mating pairs appeared on the f i r s t day,, however, pairs i n decreasing numbers were formed on succeeding days. The graph i n figure 1 follows the change i n the percentage of the population involved i n conjugation through a t y p i c a l burst. The data for the graph were obtained from dire c t counts taken at intervals during the course of the burst. The counts ranged from 730 to 1,273 c e l l s , each c e l l being scored as to whether or not i t was conjugating. From these figures an estimate was made of the percentage of the t o t a l population involved i n conjugation at the time of the count. A s i g n i f i c a n t observation from t h i s curve i s that during the four hour i n t e r v a l between the f i r s t and second counts there was approximately a 2.5 f o l d increase i n the percentage of" mating c e l l s . Equally ss s t r i k i n g i s the further observation that 24 hours e a r l i e r there were no paired c e l l s present i n the culture. When a burst of conjugation appeared i n a culture, i t did so only once and at a d e f i n i t e stage during the 30 day Figure 1. Estimated change i n the percentage of the population involved i n conjugation through a spontaneous conjugation burst. (The f i r s t count was taken as time zero.) ESTIMATED P E R C E N T OF POPULATION INVOLVED IN CONJUGATION o cn o cn o cn 12 l i f e of the culture. To demonstrate the stage at which conjugation appeared, a number of cultures of stocks CH, H, S, T and W were set up and observed daily to record the exact number of days elapsed from i n i t i a t i o n to the f i r s t appear-ance of mating pairs. Recall that these f i v e stocks belong to the group which regularly produced cultures containing large numbers of mating pairs. The results from the above experiment (shown i n Table II) indicate the burst of con-jugation begins i n the i n t e r v a l 7-24 days aft e r the i n i t i a -t i on of the culture, with an overall mean of 12.2 days. A closer look at the data reveals that 98% (123 of 126 total) of the culture dishes had the beginning of the conjugation burst f a l l i n the i n t e r v a l 7-18 days. This adjusted figure probably represents a more r e a l i s t i c i n t e r v a l than that previously stated. Under the culture conditions of t h i s investigation a prevalent observation emerges:: mating pairs rarely, i f ever, appeared i n young cultures (1-6 days) or i n older cultures (19-30 days), but pairs did appear with some regularity i n cultures which were 7-18 days old. The reasons for the appearance of mating within this r e l a t i v e l y d i s t i n c t i n t e r v a l may reside at least i n part with the n u t r i t i o n a l state of culture and will, be dealt with i n the discussion. The three numbers excluded are marked with an asterisk i n the raw data which appear i n Figure I I . 13 T a ble I I . Number o f days from the i n i t i a t i o n o f the c u l -t u r e to the f i r s t appearance o f mating p a i r s i n s t o c k s CH, H, S, T and W. CH H S T W 16 10 18 7 10 11 7 14 11 7 11 7 14 11 7 9 7 11 12 11 16 10 10 11 7 16 12 15 11 10 13 10 14 9 10 14 15 18 10 10 16 10 11 12 10 14 24* 12 12 14 14 18 12 14 11 11 14 11 16 11 11 18 11 16 14 17 10 10 14 14 12 19* 18 9 10 13 9 10 10 10 11 12 16 14 12 12 14 12 10 11 113 12 19 12 8. 20* 12 8 16 11 8 16 10 8 13 11 9 14 12 9 • 14 14 14 14 14 11 11 10 10 12 14 10 10 10 14 10 13.2 12.3 11.5 12.9 10.4 OVERALL. MEAN:- 12.2 * These f i g u r e s were excluded to g i v e the adjusted, i n t e r v a l (see t e x t f o r e x p l a n a t i o n ) . 14 2. Pre-conjugators and the Formation of Mating Pairs The accumulated observations through a great many bursts of conjugation, both i n stock cultures and i n induced bursts (induced bursts will, be discussed i n a l a t e r section), make i t possible to describe some of the features which character-ize the mating behavior of Stentor. As previously mentioned, the beginning of a burst of conjugation was characterized by the appearance of a great many mating pairs. At the same time, the early stages of a burst were marked by the appearance of numerous morphologically d i s t i n c t c e l l s which are designated as "pre-conjugator cells".. These unique c e l l s are d i s t i n c t from normal vegetative stentors i n having a portion of t h e i r l e f t f r o n t a l f i e l d and associated membranellar band folded down (.compare Figure 2a and b with c and d). Within the folded-down portion there appears a prominent bulge (see Figure 3) which eventually becomes the locus of the i n i t i a l mating union when two c e l l s j o i n to form a mating pair. Preconjugators were most evident during the f i r s t day of a conjugation burst, becoming less numerous on succeeding days. The actual mating behavior which results i n the j o i n -ing of two preconjugators to form a mating pair involves an active series of events directed toward the formation of a mating pair . Preconjugators usually remained attached to the bottom of the culture dish by t h e i r holdfasts thus leaving their "head" ends free to wave about. Two c e l l s , which were about to join, f i r s t oriented themselves i n such a way that the folded-down Figure 2. Representatives of normal vegetative individuals and pre-conjugators. (For the preconjugators the folded-down portion of the f r o n t a l f i e l d i s indicated by a bracket.) Magnification llOx. a) Ventral view of a normal c e l l . b) Anterior view of a normal c e l l . c) Ventral view of a pre-conjugator. d) Anterior view of a pre-conjugator. 16 portions of their respective f r o n t a l f i e l d s were i n close apposition (.Figure 4). Often they remained i n this position for up to f i f t e e n minutes with the c i l i a of t h e i r membranel-l a r bands touching and continually beating. Eventually the bulges on each c e l l touched and stuck, thus uniting them (Figure 5), At this point the union between the c e l l s was not.very strong for i f they were stimulated to contract (Figure 6) they often pulled apart. The folded-down portion of the f r o n t a l f i e l d was never seen to stick to any other area of any other c e l l , although having ample opportunity to do so as i t made contacts with neighbouring c e l l s , ^here-fore, the complementary stickiness appears to reside only within the folded-down portion of the f r o n t a l f i e l d and associated membranellar band, possibly only on the bulge i t s e l f . The'formation of a mating pair was always the re-sult of two pre-conjugators adhering. On no occasion were two vegetative c e l l s or a. vegetative and a- pre-conjugator seen to j o i n together. After two c e l l s became firmly united they usually re-mained r e l a t i v e l y motionless as the union between them broadened. They remained joined for varying lengths of time, usually longer than 24 hours. Figure 7 (a, b, c, d, e) shows a sequence of photomicrographs of a mating pair, tracing i t s development from i n i t i a l adhesion t i l l just before separation into two exconjugant c e l l s , a t o t a l of 28 hours. Figure 3. Anterior view of a pre-conjugator showing the bulge (indicated by the arrow) within the folded-down portion of the f r o n t a l f i e l d . Magnification llOx. Figure 4. Two pre-conjugators with the folded-down portion of their f r o n t a l f i e l d s i n close apposition; bulges not yet joined. Magnification llOx. Figure 5 . Two pre-conjugators with bulges just joined. Magnification llOx. Figure 6. Contracted c e l l s just before they p u l l apart. Magnification llOx. Figure 7. A sequence showing the development of a mating pair from i n i t i a l adhesion t i l l just before separation, a t o t a l of 28 hours. Magnification 74x. a) Bulges just united. b) 4 hours. c) 9 hours. d) 21 hours. e) 28 hours, separation came during the following hour. 19 20 3. Production of Selfing Clones The term s e l f i n g , as used by students of c i l i a t e genetics, refers i n i t s broadest sense, to the occurrence of conjuga-tion among individuals derived, by asexual reproduction, from a single parent c e l l (.i.e. intraclonal conjugation). In this respect Stentor appears to have a high tendency for s e l f i n g since six of the nine stocks regularly produced sub-cultures i n which int r a c l o n a l conjugation occurred. To further investigate the s e l f i n g a b i l i t y of Stentor, single c e l l s were isolated, placed i n separate p e t r i dish cultures and allowed to reproduce asexually. In each case, the resulting progeny were observed for the presence of intraclonal conjugation. Single c e l l s from two sources were selected: normal vege-tative stentors from each of the nine stocks and stentors from stock S which had just become paired at conjugation. For the former group a single vegetative c e l l was isolated from each stock and placed separately into a p e t r i dish c u l -ture. The source of c e l l s for the l a t t e r group requires explanation i n some d e t a i l . In order to obtain stock S c e l l s which had just become paired at conjugation i t was necessary to closely observe a culture i n the early stages of a con-jugation burst i n which there were a large number of pre-conjugators evident. When two pre-conjugators were seen to come together and stic k firmly they were quickly removed and transferred along with approximately 2 ml culture f l u i d into a small, p e t r i dish (35 x 10 mm).. Before any nuclear exchange could take place, the connection between the c e l l s 21 was severed with a f i n e glass needle. Each of the now separated c e l l s was placed into a separate p e t r i dish c u l -ture. A t o t a l of 12 c e l l s (6 s p l i t pairs) was prepared and grown i n culture by this procedure. The progress of a l l . 21 cultures produced by the two above sources was followed f o r 30 days. If , i n any of the c u l -tures, no conjugation was observed, then those dishes were subcultured and observed for an additional 30 days. This process of subculturing was repeated with each culture dish u n t i l conjugation appeared, or u n t i l f i v e subcultures had been made. The results (.Table III) show that single c e l l s from stocks CH, DF, H, S, T and W possess the a b i l i t y to produce clones which w i l l exhibit intraclonal conjugation. Further, the results show that c e l l s isolated just, after they have become paired at conjugation also have the a b i l i t y to s e l f . 4. Induced Conjugation In some cases, conjugation was found to be induced by the introduction of c e l l s belonging to one stock into a c u l -ture dish containing c e l l s of different stock. The essential feature, necessary f o r the induction, i s to mix sexually reactive c e l l s from two different stocks. Mixtures were ac-complished by the following technique. Two 12 day old pe t r i dish cultures, not yet s e l f i n g , belonging to different stocks were selected. One culture, which was to act as the donor, was placed on the stage of the stereomicroscope and the other Table I I I . Intraclonal conjugation (selfing) i n cultures started from single c e l l s Number of subcultures Presence of mating Source of single made after the pairs i n the c e l l s * i n i t i a l culture f i n a l subculture Stock C. 5 no Stock DF 0. yes Stock S 1 yes Stock SC 5 no Stock ST 5 no Stock T 3 yes Stock CH 1 yes Stock H 0 yes Stock W 3 yes c e l l a 1. yes S p l i t pair: c e l l b died c e l l a 2 yes S p l i t pair: celL b 0 yes c e l l a died S p l i t pair: c e l l b 2 yes c e l l a 1 yes S p l i t pair: c e l l b 1 yes c e l l a 3 yes S p l i t pair: c e l l b 1 yes c e l l a 1 yes S p l i t pair: c e l l b 1 yes * A l l . s p l i t pairs were from stock S.. 23 beside the microscope., With the aid of the microscope and using a Pasteur pipet, approximately 300 stentors were carefully removed from the agar surface of the donor c u l -ture dish and introduced into the other culture dish. Subsequent observation of the recipient culture dish revealed a s t r i k i n g sequence of events which w i l l be referred to as the mating reaction. After a refractory period l a s t i n g about 5^ -7 hours there was the gradual appearance of numerous pre-conjugator c e l l s . This was followed immediately by the form-ation of mating pairs. The reciprocal experiment, i n which the donor stock culture of the previous experiment became the recipient of c e l l s from the extraneous stock, showed a similar mating reaction, again leading to the formation of mating pairs. The induced conjugation bursts were different, i n some respects from the bursts of conjugation which oc-curred spontaneously i n p e t r i dish cultures of the nine stocks. F i r s t , there were f a r more pre-conjugators found i n the i n -duced conjugation bursts. Pre-conjugators numbering i n excess of 100 were frequently seen at one time during induced conjugation bursts, whereas, i n spontaneous bursts usually less than forty pre-conjugators were evident at any one time. Secondly, i t was possible, i n the induced bursts, to observe many mating pairs being formed as their bulges adhered, while i n spontaneous bursts i t was d i f f i c u l t to fi n d mating pairs just as they were being formed. This difference i s probably due to the greater number of pre-conjugators being present i n the induced conjugation burst. 24 5. Specific Mating Between Stocks Early i n the investigation i t was found that not a l l combinations of c e l l s from the various stocks gave the induced mating reaction. In order to determine which combinations would result i n an induced conjugation burst, the stocks were 2 mixed together i n a l l possible combinations of twos . To ac-complish t h i s , each stock was tested i n d i v i d u a l l y by adding c e l l s from the remaining seven different stocks (donors) to a series of p e t r i dish cultures (recipients) of the stock being tested« The control i n each case was made by adding c e l l s which belonged to the stock being tested, to a culture of that same stock. The mixtures were conducted i n the man-ner previously described for the induction of conjugation. Each mixture was subsequently observed for the appearance of an induced conjugation burst. A mixture was assumed to be lacking i n any capacity to give a mating reaction i f after 24 hours no pre-conjugators or mating pairs were formed. The results (Table IV) show the stocks to be d i v i s i b l e into two groups with respect to their mating r e a c t i v i t y i n mix-tures: group I consists of stocks C, SC and ST,, while group II consists of stocks S, T, CH, H and W. No, mating reaction was observed i n mixtures of c e l l s of two stocks belonging to the same group. But, a mating reaction did occur i n a l l mix-tures where c e l l s from any stock i n group I were combined Stock DF was not included i n thi s experiment because i t was not obtained u n t i l the l a t t e r part of the investigation. 25 Table IV. Specific mating i n mixtures of stocks. Donor stocks C SC ST S T CH H W Stocks tested (recipients)* C SC ST S T CH H + + + + + + + •f-+ + + +• + + + W + + + + * Each v e r t i c a l column represents a series of cultures of a single stock which were tested f o r t h e i r a b i l i t y to give a mating reaction with c e l l s from the donor stocks. A plus (+) sign indicates a mating reaction occurred. A minus (-) sign indicates no mating reaction occurred. with c e l l s from any stock i n group I I . These results strongly suggest that the formation of mating pairs i n appropriate mixtures i s the direct result of the pre-sence of two complementary mating types. I t , therefore, seems reasonable to tentatively assign mating types to group I and II.. Hence,, the stocks i n group I become mating type I and those i n group I I become mating type I I . 6. Demonstration of Cross-mating It was desired to determine whether the mating observed during induced conjugation bursts was i n the form of cross-mating, with one c e l l of a pair belonging to one stock and the other c e l l belonging to the other stock. Several preliminary observations led to the early b e l i e f that cross-mating did occur during induced conjugation bursts. These came from the scrutiny of individual mating pairs formed i n mixtures; i n the majority of pairs, the two c e l l s were recognizably di f f e r e n t with respect to size and inten-s i t y of pigmentation, r e l a t i v e to each other. Figure 8 shows a characteristic mating pair formed i n a mixture of stock C and stock S c e l l s . The larger more darkly pig-mented c e l l almost certainly belongs to stock S, whereas the smaller, more l i g h t l y pigmented c e l l almost certainly belongs to stock C. This judgement was reinforced by measuring the diameter of contracted c e l l s of both stocks; measurement revealed a difference i n size such that c e l l s of stock S averaged 272 microns while c e l l s of stock C were 27 smaller, averaging only 220 microns. Further, when c e l l s from stocks S and C were placed together i n a drop of water on a s l i d e and observed with the stereomicroscope, the d i f -ference i n pigmentation intensity, although not great, was apparent. Labelling with carmine was used to investigate the occurrence of cross mating more f u l l y and to obtain quan-t i f i a b l e r e sults. Labelled c e l l s of a group I stock were mixed with unlabelled c e l l s of a group II stock and both c e l l s of the res u l t i n g mating pairs were ind i v i d u a l l y scored on the basis of three distinguishing characteristics: presence or abs.ence of l a b e l , l i g h t or dark pigmentation with respect to each other, and large or small size with respect to each other. For the experiment, stocks C and S were chosen because the difference between them, with respect to size and pigmentation,was most easily seen. Labelling was accomplished by the introduction of a carmine p a r t i c l e suspension (prepared by suspending 0.1 g powdered carmine i n 1 ml culture f l u i d ) into a culture con-taining stock G. Immediately, the c e l l s began to ingest the carmine and after 15 minutes their cytoplasms contained large quantities of the l a b e l . Following t h i s treatment, approximately 300 newly labelled c e l l s were removed, washed by centrifugation i n three changes of fresh medium to remove any free carmine p a r t i c l e s , and then added to a p e t r i dish culture containing stock S. Twelve hours l a t e r the r e s u l t -ing mating pairs were removed, placed one at a time on a glass s l i d e i n a drop of culture f l u i d , and separated with a f i n e glass needle. Each c e l l of the pair was then placed i n a separate drop of culture f l u i d on the sl i d e , squashed l i g h t l y with a cover glass (Figure 9 ) , and examined f o r the presence of carmine p a r t i c l e s using the steromicroscope. Altogether, three separate experiments were performed with stocks C and S using the above l a b e l l i n g technique. The results (Table V) for each mating pair were scored on the basis of pigmentation, size and presence of carmine, re-sulting i n the emergence of six classes of mating pairs from the combined results of a l l three experiments. How-ever, a close examination of the data i n Table V revealed that some classes may be amalgamated to give a t o t a l of three d i s t i n c t classes: f i r s t , a cross-mating class (com-bining classes 1, 2 and 3) i n which mating pairs consist of one stock C c e l l and one stock S c e l l ; secondly, a s e l f i n g class (combining classes 4 and 5) i n which mating pairs consist of two stock G c e l l s ; and t h i r d l y , an additional s e l f i n g class (class 6 only) i n which mating pairs consist of two stock S c e l l s . This r e d i s t r i b u t i o n i s based on the probability that some of the labelled stock C c e l l s had extruded the foreign carmine p a r t i c l e s thus leaving them unlabelled. This assumption i s supported by the presence, after 12 hours, of carmine p a r t i c l e s on the bottom of the culture dish where at the beginning of the experiment there had been none. Figure 8. Mating pair from a mixture of stocks C and S. Magnification 104x. Figure 9. Squashed pair from a mixture of labelled stock S c e l l s and unlabelled stock C c e l l s . (The arrows indicate the carmine p a r t i c l e s . ) Magnification 104x. Table V. Observed Classes of Mating Pairs Scored on the Basis of Relative Pigmentation, Presence of Carmine, and Relative Size. S p l i t pair ° C e l l a C e l l b Number of mating pairs class of p a i r pigment carmine size pigment carmine size Expt 1 Expt 2 Expt 3 Total each class 1 l g t + sml drk - l r g 33 29 41 103 2 Igt -* sml drk l r g 4 2 4 10 3 l g t + sml drk ' l r g 0 1 0 ' 1 4 l g t sml l g t + sml 1 1 1 3 5 l g t + sml l g t sml 0 0 1 1 6 drk - l r g drk - l r g _1 _0 _1 2 Totals: 39 33 48 Total pairs sampled: 120 Symbols and abbreviations: + carmine p a r t i c l e s present; - no carmine p a r t i c l e s present; l g t = l i g h t l y pigmented c e l l ; drk =darkly pigmented c e l l ; sml =smaller c e l l ; l r g = l a r g e r c e l l * The absence of carmine p a r t i c l e s i n these c e l l s i s probably due to the c e l l s having extruded the foreign p a r t i c l e s (see text f o r a further explanation). * This c e l l contained a single very small carmine p a r t i c l e which was probably picked up from the bottom of the culture dish'. 31 The redistributed results strongly suggest that 95% of the mating pairs formed i n the mixture were of the cross-mating type, while the remaining 5% were of the s e l f i n g type. The above l a b e l l i n g experiments were performed only with mixtures of c e l l s from stocks G and S. However, i t i s s i g n i f i -cant that i n mixtures of other stocks, which resulted i n an induced conjugation burst, the majority of m&ting pairs formed appeared to be of the cross-mating type, judged on the observed differences i n size and pigmentation of the two c e l l s making up the pairs. The high incidence of cross-mating overwhelmingly supports the conclusion that the mating reaction, which occurred only i n certain mixtures,was due to the presence of complementary mating types ( i . e . mating types I and I I ) . 7. The Role of C e l l to C e l l Contacts i n the Induction of  Conjugation The actual stimulus which caused the mating reaction when c e l l s from two different stocks were mixed was not obvious, but, two d i s t i n c t p o s s i b i l i t i e s were considered. F i r s t l y , the c e l l s of either or both stocks may have put some factor into the medium which caused other c e l l s to become pre-conjugators thus leading to the observed mating reaction. The second p o s s i b i l i t y (suggested by Sonneborn i n a personal communication) i s that the mating reaction may have been a consequence of a "contact interaction" between sexually reactive c e l l s . Such contact phenomena 32 have been found to exist i n Euplotes (Heckman and Siegel, 1964) where i t has been shown that the c e l l s i n a mixture of two mating types actually have to make contacts, not contacts which result i n sticking together, but simply make and break contacts which stimulate the c e l l s to become ready for conjugation some hours l a t e r . In order to determine the cause of the mating reaction i n Stentor, an experiment was performed i n which a series of three d i f f u s i o n chambers was used to i s o l a t e groups of c e l l s . The central idea was to prevent the isolated c e l l s from contacting c e l l s of another mating type, while, at the same time, subjecting them to the influence of any d i f f u s i b l e factors which might cause a mating reaction. The d i f f u s i o n chambers (Figure 10a, b) were made from 2 cm lengths of slotted plexiglas tubing (one inch i n diameter) wrapped with nylon bolting cloth having a pore size of 62 microns (obtained from John Staniar & Co., Sherborn Street, Manchester, England). The three chambers (lettered A, B and C i n Figure 11) were placed into a 12 day p e t r i dish culture of stock C, each chamber i n an area which had previously been cleared of c e l l s . The chambers were pressed firmly into the agar layer thus e f f e c t i v e l y seal-ing the bottom of the chamber. Diffusion chambers pre-pared i n th i s manner prevented the passage of stentors either into or out of the chamber, but did not prevent the passage of the food organisms which were about 30 microns i n length. Figure 10. Diffusion chamber (a) before wrapping with bolting cloth and (b) aft e r wrapping i s complete. 33 Figure 1 1 . P e t r i dish culture with d i f f u s i o n chambers i n place. 34 To i n i t i a t e an experiment, c e l l s belonging to stocks C and S were al l o t t e d to the three chambers i n the following way: chamber A received 100 stock C c e l l s , chamber B received 100 stock S c e l l s , and chamber C received 50 stock C c e l l s plus 50 stock S c e l l s . Addition-a l c e l l s of stock S were added to the p e t r i dish i n the area outside of the d i f f u s i o n chambers. The procedure described so f a r represented one half of a two part ex-periment with both parts being executed concurrently. The other half.of the experiment was the reciprocal of the part just described and was performed i n the same manner except that the d i f f u s i o n chambers were placed into a p e t r i dish culture of stock S instead of stock C. Both culture dishes were subsequently observed for the presence of pre-conjugators or mating pairs. After seven hours there were, i n both dishes, pre-conjugators and mating pairs present i n chamber C and i n the area outside the d i f f u s i o n chambers; both of these areas contained c e l l s from stocks C and S mixed together. At the same time, chamber A (containing only stock C c e l l s ) and chamber B (containing only stock S c e l l s ) showed no evidence of a mating reaction with no pre-conjugators or mating pairs being formed even after 24 hours. This experi-ment was repeated three times with the same re s u l t s . The results of th i s series of experiments indicate a lack of any d i f f u s i b l e factor affecting the mating a b i l i t y of c e l l s . I t , therefore, seems evident that the only d i f -ference between c e l l s i n chambers A or B and those i n the 3 6 other areas, l i e s i n the i n a b i l i t y of the c e l l s contained i n chambers A and B to make contacts with c e l l s of a diffe r e n t mating type. Further evidence suggesting the absence of any d i f f u s i b l e factor acting i n the induction of conjugation was found i n the i n a b i l i t y of conditioned medium taken from actively conjugating cultures to induce conjug-ation when added to a non-conjugating culture from which most of the f l u i d had been drained. This was repeated several times using different cultures with no apparent effect of culture f l u i d upon c e l l s . These results strongly suggest that contacts between c e l l s belonging to diff e r e n t mating types play an essential role i n the induction of conjugation. 8. Survival of Exconjugants Although an extensive study of exconjugants was not made, the dominant impression acquired from the follow up of ex-conjugants was the i r apparent high mortality rate. The high mortality i s evident after a conjugation burst when large numbers of sluggish, poorly pigmented c e l l s appear which l i v e only ;a few days. In several attempts to obtain l i v i n g exconjugants, a t o t a l of 80 mating pairs was isolated from different c u l -tures at various times during the course of the investiga-ti o n . In each case the mating pairs were placed separately into small p e t r i dishes containing new medium and food organ-isms. After a pair separated, one c e l l was transferred to 37 another small p e t r i dish containing new medium and food organisms. Of the t o t a l of 160 exconjugants produced by the separation of the 80 pairs, only two c e l l s , derived from different pairs isolated from a conjugation burst i n stock W, survived long enough to begin d i v i s i o n . These two surviving exconjugants produced clones which were subcultured continuously for six months, however, no mating pairs were observed i n either l i n e of descent. The remain-ing 158 exconjugants died within a few days afte r i s o l a t i o n . The most successful attempts to keep exconjugants a l i v e were with large groups of mating pairs which were isolated and placed into a p e t r i dish culture. Four groups of mating pairs, ranging i n size from 10 pairs to 60 pairs, were i s o -lated and placed into new p e t r i dish cultures. One group came from a conjugation burst i n a stock T culture and the other three groups came from induced conjugation bursts i n mixtures of stocks C and S. Although a large number of the exconjugants i n these groups died, a few did survive i n each dish, eventually giving r i s e to a population. After 30 days i n culture, during which no conjugation was observed, a l l four dishes were subcultured. The population which descended from the stock T exconjugants showed conjugation within the f i r s t subculture. This was also true i n two of the three lines of descent which originated from the mixtures of stocks C and S. The remaining l i n e did not show any conjugation after four subcultures. To b r i e f l y summarize, i n the presence of a high mortality 38 rate some exconjugants did survive and divide; i n some cases they gave r i s e to a population of c e l l s which con-jugated i n the f i r s t subculture, less than 60 days after ; their previous mating. 9. Abnormal Mating During the course of the investigation, two types of abnormal mating were observed. F i r s t , a small number (approximately 15) of mating c e l l s showing "multiconjugation" were observed i n which three c e l l s were joined together i n -stead of the usual two. Figure 12 shows an example discov-ered i n a conjugation burst induced by a mixture of c e l l s from stocks C and S. A second type of abnormal mating, found i n a stock S culture, was seen i n a single mating pair i n which one of the c e l l s was at a late stage of d i v i -sion (Figure 13). The dividing c e l l appeared to be at stage 7 (Tartar, 1961) and remained at that stage throughout the four hour period during which i t was observed. Abnormal mating, of the two types described, appeared infrequently throughout the investigation. Figure 12.. An example of a mating t r i p l e t found i n a mixture of stocks C and S. (The two larger c e l l s appear to be stock S; the smaller c e l l probably belongs to stock C.) Magnification l l O x . Figure 13. Mating pair with one partner at about stage 7 of d i v i s i o n . Magnification llOx. 39 40 DISCUSSION In many c i l i a t e s , conjugation i s most readily induced by mixing cultures of different o r i g i n . From the results of these mixtures, the cultures can be c l a s s i f i e d into two or more mating types, being classed as complementary mating types i f conjugation i s induced by the mixture (Sonneborn, 1957). By this d e f i n i t i o n individuals belonging to the same mating type do not conjugate (except i n certain cases of s e l f i n g ) . But when individuals of two different mating types are mixed they w i l l unite. Mating types have been found i n seven genera of c i l i a t e s ; namely, Colpidium, Euplotes, Oxytricha, Paramecium, Tetrahymena, Sty1onychia, and Tokophyra (Sonneborn, 1957; Allen, 1967). After the i n i t i a l recurrent observations of conjugation i n stock cultures i t seemed reasonable to begin a search for mating types i n Stentor coeruleus. The success of the search was enhanced by the discovery of a method to induce conjugation, i n which the essential feature was the mixture of c e l l s from two diverse stocks. It became apparent from mixture experi-ments that the eight stocks tested represented two comple-mentary mating types on the basis of their response when mixed, two at a time, i n a l l possible combinations (see Table IV). It was, therefore, possible to assign stocks to either mating type I or mating type I I . This d i v i s i o n of stocks into mating types was d i s t i n c t i n that mixtures consisting of c e l l s of the same mating type did not result 41 i n a mating reaction. But, when c e l l s of diverse mating type were mixed, a mating reaction occurred. By th i s d e f i n i t i o n , stocks C, SC, and ST belong to mating type I and react i n a p a r a l l e l manner when mixed with stocks belonging to mating type I I . In a similar way, stocks CH, H, S, T and W belong to mating type II and react i n a p a r a l l e l manner when mixed with mating type I stocks. The occurrence of two mating types i n the present study should not be taken to imply that only two mating types exist i n this species. In several c i l i a t e species, which have been studied extensively, rather complex systems of i n t e r -breeding mating types have been recognized. In these, a particular species may have either a single variety with two or more interbreeding mating types (e.g. Euplotes  crassus Heckman, 1964) or several v a r i e t i e s , each with two or more interbreeding mating types (e.g. Paramecium  aurelia, Sonneborn, 1957). Interbreeding between individuals of different v a r i e t i e s does not occur. By this interpreta-tion, the eight stocks of the present study belong to the same variety since they constitute two interbreeding mating types. However, the small number of genetically d i s t i n c t stocks (probably f i v e ; see Appendix) used i n this investiga-tion emphasizes i t s preliminary nature. It seems reasonable, therefore, to suppose that future studies, using larger numbers of stocks collected from widely distributed areas, w i l l reveal additional mating types and possibly d i f f e r e n t v a r i e t i e s . 42 In most c i l i a t e s , mating pairs formed i n mixtures of two complementary mating types always consist of individuals of different mating type. There have, however, been a few cases reported where a small f r a c t i o n of the pairs formed i n mixtures was apparently due to the union of c e l l s of the same mating type (Hiwatashi, 1951; Metz, 1954; Katashima, 1961; Larison and Siegel, 1961). Pairs formed following mixtures of diverse cultures of Stentor regularly consist of c e l l s of diverse types. This conclusion i s based on the results of the l a b e l l i n g experiments i n which the majority of pairs consisted of one labe l l e d end one unlabelled c e l l (Table V). The explanation for the occurrence of the few pairs which appeared to be i n the form of s e l f i n g i s not clear. Metz (1954) showed that i n Paramecium c e l l s belonging to a single clone can mate amongst themselves after some of the individuals- have made transient contacts with c e l l s of a complementary mating type. The explanation Metz offered was that c e l l s which have been temporarily united with i n d i v i d -uals of a complementary mating type may i n some cases acquire that mating type s p e c i f i c i t y i n the course of the contact. Thus a transitory s h i f t of mating type may occur i n these cases, resulting i n the formation of s e l l i n g pairs. Hiwatashi (1951) found that s e l f i n g occurred i n f i v e per-cent of mating pairs formed i n mixtures of Paramecium  caudaturn. In Stentor, s e l f i n g i s also evident i n the occurrence of conjugation i n some stock cultures and clonal cultures. 43 This type of s e l f i n g probably has a different basis i n i t s spontaneous appearance than that which appeared i n mixtures. The appearance of the majority of spontaneous conjugation bursts during the in t e r v a l 7-18 days afte r the i n i t i a t i o n of the cultures (see Table II) seems best explained as being due to the n u t r i t i o n a l state of the culture. The n u t r i t i v e state of the protozoan seems to be an important factor influencing conjugation i n a l l c i l i a t e s . Sonneborn (1939) observed that i n Paramecium aurelia the mating reaction does not take place i n cultures that are either over-fed or completely starved. A similar s i t u a t i o n exists i n P. caudatum (Gilman, 1939) and P. bursaria and P. ca l k i n s i (Wichterman, 1953). Geise (1939) found that food appeared to be the most important single factor i n regard to conjugation i n P. multimicronucleatum and that a decline i n available food supply after a period of plenty was required. In practice, c i l i a t e s are brought into mating condition f o r conjugation studies by subjecting them to a declining n u t r i -t i o n a l regime (Sonneborn, 1957; Allen, 1967). Stentors cultured i n the manner described are i n i t i a l l y provided with a period of n u t r i t i o n a l abundance during which they multiply rapidly. But, as they approach maximum density the food organism population dwindles and the stentors enter a period of famine. It i s during the t r a n s i t i o n a l period, when the food population i s declining that the burst of conjugation appears. Therefore, Stentor appears to respond to a decline 44 i n food supply after a period of plenty by s e l f i n g . The fact that Stentor s e l f s brings up a problem i n regard to i t s mating type system. The problem l i e s i n the explanation of the basis of pair formation i n a population which has not been mixed with another mating type. Selfing i n pure cultures i s not uncommon i n c i l i a t e s i n which mating types have been found to exist. The s e l f i n g conjugations previously reported f o r Paramecium bursaria (Jennings, 1941) P. aurelia (Kimball, 1939a; Sonneborn, 1947), P. multimicro-nucleatum (Sonneborn, 1957), Tetrahymena pyriforalis , ('Nanneyl and Chaughey, 1955) and Euplotes crassus (Heckman, 1964) always resulted from the d i f f e r e n t i a t i o n of individuals of complementary mating type within the s e l f i n g culture. In these cases, the s e l f i n g pairs were formed as e result of the union of c e l l s of complementary mating, type. In the few cases i n which such intraclonal mating has been studied i t was explained as either due to macronuclear heterogeneity and assortment of macronuclear subunits during clonal expan-sion (Allen and Nanney, 1958), or as a consequence of variable gene .expression (Butzel, 1955). Recent studies have shown the l a t t e r to be the most l i k e l y interpretation. Heckman (1964, 1967) found i n Euplotes crassus that s e l f i n g i n old heterozygotes was due to a normally recessive a l l e l e being expressed. In Paramecium aurelia. s e l f i n g , interpret-able as due to changes i n gene a c t i v i t y , has been extensively studied (Sonneborn, 1966; Taub, 1966; and Bleyman, 1967a). Returning, now, to the influence of n u t r i t i o n on conjugation, 45 Hiwatashi (1958) found that s e l f i n g i n Paramecium caudatum was susceptible to environmental (nutritional) control. Mating type changes were influenced by varying growth conditions, high f i s s i o n rate favored one type, low another (Hiwatashi, 1960). Bl eyman (1967b) found that i n Paramecium  aurelia a period of rapid growth at 27° C followed by a depletion of food at 19° C would bring about maximal rea c t i v -i t y , both for s e l f i n g and mating with standard testers. Both- Hiwatashi and Bleyman observed that t h e i r respective s e l f i n g stocks tended to produce cultures which consisted predominantly of one mating type, but that the cultures ranged from those which did not s e l f to those which regularly selfed, depending upon their o r i g i n and culture conditions. This observation i s reminiscent of the condition which i s found i n the s e l f i n g stocks of Stentor. It i s interesting that a l l the stocks which s e l f regularly belong to mating type II, while those which only rarely s e l f belong to mating type I. Gilman (1941) si m i l a r l y reported that i n Paramecium caudatum s e l f i n g i s common i n some v a r i e t i e s and i t may occur more often i n cultures of one prevailing mating type than i n cultures of the other. In view of the findings reported for other c i l i a t e s , s e l f -ing i n Stentor may be due to a persistent i n s t a b i l i t y of mating type expression such that transitory changes i n mating type would lead to the situation where two complementary mating types were present i n a culture. Further, the cultures 46 may be predominantly of one mating type, but a period of rapid growth followed by a depletion of food may cause some individuals to temporarily change mating type, thus permitting the formation of mating pairs. This would explain the appear-ance of s e l f i n g after 7-18 days i n culture. Depending upon the o r i g i n of the culture the mating type changes may involve type I expressing type II or alternatively, type II expressing type I. However, the d i s t i n c t p o s s i b i l i t y that future investiga-tions may reveal additional mating types capable of interbreed-ing with the present types makes i t necessary to consider the p o s s i b i l i t y that individuals of type I or type II may be able to express a mating type not yet discovered, a situation which would result i n s e l f i n g . It was possible to determine the mating type of the s e l f i n g stocks by choosing cultures which had not begun to s e l f , presumably containing c e l l s ex-pressing only one mating type. When mating types I and II of Stentor were brought to-gether under conditions which were favorable f o r conjugation, the onset of mating behavior occurred only after a waiting period of 5-7 hours. The waiting period (often celled the refractory period) i s apparently widespread among the c i l i a t e s for i t has been reported for Euplotes (Kimball, 1939b; Katashima 1959; Heckman 1963, 1964), Stylonychia (Gr e l l , 1951; Downs, 1952) Oxytricha (Siegel, 1956), and Tetrahymena ( E l l i o t and Gruchy, 1952; Nanney and Caughey, 1953). In mixtures of complementary mating types i n Euplotes. i t has been shown that during the waiting period repeated c e l l contacts are 47 made between the two mating types, leading subsequently to formation of mating pairs (Heckman and Siegel, 1964). The hypothesis here was that the contacts bring about an exchange of information which causes the c e l l s to become ready for conjugation some hours l a t e r . Heckman and Siegel provide support f o r their hypothesis by demonstrating that the wait-ing period can be eliminated i f c e l l s of different mating types are each permitted to make previous contact with c e l l s of a third mating type. Thus, by pre-treating the c e l l s they found that when the mixture was made, mating occurred immediately between the f i r s t two types. In Stentor, c e l l to c e l l contacts appear necessary f o r the i n i t i a t i o n of the mating reaction. In addition, there appears to be a lack of any d i f f u s i b l e factor which might affect the mating a b i l i t y of c e l l s . When c e l l s of either mating type I or type II were placed into a d i f f u s i o n chamber i n such a way that they could not contact c e l l s of the other mating type but at the same time were subjected to the environ-mental influences of the other mating type, they did not form pre-conjugators or mating pairs. In sharp contrast, when c e l l s of mating type I and II were placed together into a di f f u s i o n chamber the normal waiting period was followed by the appearance of pre-conjugators and, subsequently, mating pairs. The results of these experiments provide powerful evidence i n support of the hypothesis that contact between c e l l s of different mating type provides a stimulus which causes the c e l l s to become ready for conjugation some hours 48 l a t e r . The nature of the information (stimulus) which appears to be exchanged i s unknown. Metz (1954) presented an hypo-thesis which holds that c e l l unions leading to conjugation are brought about by the interaction of complementary mating type substances which he said were protein i n nature. Evidence indicating that protein synthesis i s necessary for the appearance and maintenance of mating a b i l i t y i s provided by Bleyman (1964) for Paramecium aurelia and by Cohen (1965) for P. bursaria. It has been suggested (Heckman and Siegel, 1964) that upon i n i t i a l contact c e l l s of complementary meting type cannot unite but molecular amounts of mating type sub-stances could be exchanged. Then, the "foreign" protein could stimulate synthesis of additional mating type sub-stances so that i n due course the c e l l u l a r concentrations would be s u f f i c i e n t l y high to allow c e l l unions. The wait-ing period would, of course, be the time during which the new mating type substances were being synthesized. Stentor seems to respond to contact with c e l l s of comple-mentary mating type i n a manner which i s at least p a r t i a l l y similar to that suggested for Euplotes by Heckman and Siegel. But, Stentor shows an additional response i n the folding down of a portion of i t s l e f t f r o n t a l f i e l d as i t takes on the form of a pre-conjugator. I f contacts with c e l l s of another mating type stimulates synthesis of new mating type substances i n this genus, then such synthesis apparently 49 results i n a highly l o c a l i z e d build up of such substances. The i n i t i a l union at conjugation has i t s locus solely with-in' the folded-down portion of the fr o n t a l f i e l d , possibly only on the bulge produced within i t . Also, synthesis i s evidently not completed u n t i l the c e l l has taken on the form of a pre-conjugator, since contacts previous to that time do not result i n c e l l unions. F i n a l l y , the observed high mortality of exconjugants produced i n s e l f i n g cultures may be due to inbreeding degen-eration resulting from the accumulation of deleterious recessive genes i n homozygous condition. Nanney (1956, 1957) found that during the course of inbreeding i n strains of Tetrahymena various signs of inbreeding degeneration appeared, one of which was death at conjugation. Also, there i s good evidence to indicate that the use of old stocks (clones) may result i n a general non-viability among exconjugants CSiegel, 1967). The stocks employed i n the present study have i n some cases been cultured f o r periods i n excess of ten years. A large portion of the age-correlated mortality i n c i l i a t e s seems to be due to progressive deterioration of micronuclei. Several instances of chromosomal i r r e g u l a r i -t i e s i n old stocks have been reported f o r Paramecium .(Bippell, 1955; Sonneborn and Schneller, ,1960) and Tetra-hymena (Nanney and Nagel, 1964; Wells, 1965). A high mortality among exconjugants of Stentor has also been encountered by B u r c h i l l (1967) and by Harden and Holland (1968). However, both of these studies report the 50 survival of some exconjugant c e l l s , as does the present study. These viable exconjugants appeared quite normal, and were able to p r o l i f e r a t e , eventually producing clones. The fact that some exconjugant c e l l s do survive i s encourag-ing and should serve as a stimulus to seek the conditions necessary to increase v i a b i l i t y . In retrospect, the reported occurrence of mating types i n Stentor should provide an experimental system with which to investigate inheritance i n thi s genus. In addition, i f conjugation can be brought under precise and stringent experimental control then biology would be provided with a most powerful tool toward analyses of very fundamental ques-tions. Cross breeding could be combined with microsurgery so that one could ask, for example, whether the macronucleus from a mating type I c e l l , when transplanted to a mating type II c e l l , w i l l cause the host c e l l now to become mating type I - again, w i l l cytoplasm from a mating type I c e l l plus macronucleus from a mating type II. c e l l give, i n combin-ation, a mating type II c e l l . The outcome of such experiments would provide valuable information on the relationships which exist between the nucleus and the cytoplasm. SUMMARY 51 1. Two experimental methods, which regularly y i e l d large numbers of mating pairs were used to investigate conjugation i n Stentor coeruleus. One method involves a particular culture technique, the other requires a mixture of different stocks. 2. Mating pairs appeared i n the form of conjugation bursts, either induced by mixing certain stocks, or occurring spontaneously i n some stock cultures and i n clones produced by single c e l l s isolated from them. 3. Spontaneous conjugation bursts, i n the majority of cases occurred during a def i n i t e i n t e r v a l i n the development of a culture, possibly due to the n u t r i t i o n a l conditions within the culture. 4. Morphologically d i s t i n c t pre-conjugator .cells appear immediately before as well as during the i n i t i a l stages of a conjugation burst. Mating pairs are formed by the union of pre-conjugators. 5. Mixing eight stocks i n a l l possible combinations of twos and observing t h e i r subsequent response revealed they were separable into two complementary mating types. 6 . The majority of mating pairs formed i n mixtures were of the cross-mating type. 7. Contact between c e l l s of d i f f e r i n g mating type appears to be necessary for the i n i t i a t i o n of a mating reaction. 52 BIBLIOGRAPHY Allen, S.L., 1967. Chemical Genetics of Protozoa. In "Chemical Zoology." Vol. I. Academic Press. New York pp. 617-694. Allen, S.L., and D.L. Nanney, 1958. Analysis of nuclear dif f e r e n t i a t i o n , i n sel f e r s of Tetrahymena. Amer. Naturalist 92: 139-160. Balbiani, E.G., 1891. Novelles recherches experimentales sur l a merotomie des infusoires c i l e s . Annales Micrograph. 4: 369-407. Bleyman, L.K., 1964. The i n h i b i t i o n of mating r e a c t i v i t y i n Paramecium aurelia by i n h i b i t o r s of protein and RNA synthesis. Genetics 50: 236 (abstract). Bleyman, L.K., 1967a. Selfing i n Paramecium aurelia, Syngen 5:: Persistent i n s t a b i l i t y of mating type expression. J . Exptl. Zool. 165: 139-146. Bleyman, L.K., 1967b. Determination and inheritance of mating type i n Paramecium aurelia syngen 5. Genetics 56: 49-59. B u r c h i l l , B.R., 1967. Conjugation i n Stentor coeruleus. J . Proto. 14: 683-687. Butzel, H.M., 1955. Mating type mutations i n variety I of Paramecium aur e l i a and t h e i r bearing upon the pro-blem of mating type determination. Genetics 40: 321-330. Cohen, L.W., 1965. The basis f o r the circadian rhythm of mating i n Paramecium bursaria. Exptl. Cell.Res. 37: 360-367. De Terra, N., 1966. Culture of Stentor coeruleus on Colpidium campylum. J . Proto. 13: 491-492. Dippell, R.V., 1955. Some cytological aspects of aging i n variety 4 of Paramecium a u r e l i a . J . Proto. 2: 7 (abstract). Downs, L.E., 1952. Mating types i n Stylonochia putrina. Proc. Soc. Exp. B i o l . Med. 81: 605-607. E l l i o t , A.M., and D.E. Grunchy, 1952. The occurrence of mat-ing types i n Tetrahymena. B i o l . B u l l . 103: 301 (abstract). Geise, A.C, 1939. Studies on conjugation i n Paramecium multimicronucleatum. Amer. Naturalist 73: 432-444. 53 Gilman, L.C., 1939. Mating types i n Paramecium caudatum. Amer. Naturalist 73: 445-450. Gilman, L.C, 1941. Mating types i n diverse races of Paramecium caudatum. B i o l . B u l l . 80: 384-402. G r e l l , K.G., 1951. Die Paarungreaktion von Sty1onychia  mytilus. Zeitschr. Naturforsch. 66.: 45-47. Hamburger, C , 1908. Zur Kenntnis der Conjugation von Stentor coeruleus nebst einigen allgemeinen Bemerkungen uber die Conjugation der Infusorien. Z. Wiss Zool. 90: .421-433. Harden, CM., and T. Holland, 1968. The effects of conjuga-tion i n Stentor coeruleus. J . Proto. (In press). Heckman, K., 1963. Paarungssystem und genabhangige Paarung-stypdifferenzierung bei dem hypotrichen C i l i a t e n Euplotes vannus. Arch. Protistenk. 106: 393-421. Heckman, K., 1964. Experimentelle Untersuchungen an Euplotes crassus. I Paarungssystem, Knojugation and Determination der Paarungstypen. Z. Vererbunysl. 95: 114-124. Heckman, K., 1967. Age-dependent intraclonal conjugation i n Euplotes crassus. J . Exptl. Zool. 105: 269-278. Heckman, K., and R.W. Siegel, 1964. Evidence for the induc-ti o n of mating-type substances by c e l l to c e l l contacts. Exptl. C e l l Res. 36: 688-691. Hiwatashi, K., 1951. Studies on the conjugation of Paramecium  caudatum IV. Conjugating behavior of individuals of two mating types marked by a v i t a l staining method. S c i . Rep. Tohoku Univ. (Biol.) 19: 95-99. Hiwatashi, K., 1958. Inheritance of mating types i n variety 12 of Paramecium caudatum. S c i . Rep. Tohoku Univ. (Biol.) 24:; 119-129. Hiwatashi, K., 1960. Analysis of the change i n mating type during vegetative reproduction i n Paramecium caudatum. Jap. J . Genetics 35: 213-221. Jennings, H.S., 1941. Genetics of Paramecium bursaria. I I . S e l f - d i f f e r e n t i a t i o n and s e l f - f e r t i l i z a t i o n of clones. Proc. Amer. phil.- Soc. 85: 25-48. Katashima, R., 1959. Mating types i n Euplotes eurystomus. J . Proto. 6: 75-83. 54 Katashima, R., 1961. Breeding system of Euplotes pat e l l a i n Japan. Jap. J . Zool. 13: 39-61. Kimball, R.F., 1939a. Change of mating type during vegetative reproduction i n Paramecium aure l i a. J . Exptl. Zool. 81: 165-179. Kimball, R.F. 1939b. Mating types i n Euplotes. Amer. Naturalist 73: 451-456. Larison, L.L., and R.W. Siegel, 1961. Illegitimate mating i n Paramecium bursaria and the basis for c e l l union. J . gen. Microbiol. 26: 499-508. Metz, C.B., 1954. Mating substances and the physiology of f e r t i l i z a t i o n i n C i l i a t e s . In "Sex i n Microorganisms". Symp. Amer. Assoc. Advanc. S c i . pp. 284-334. Moxon, W., 1869. On some points i n the anatomy of Stentor and on i t s mode of d i v i s i o n . J . Anat. Physiol. 3: 279-293. Mulsow, W., 1913. Die Conjugation von Stentor coeruleus und Stentor polymorphus." Arch. Protistenk. 28: 363-388. Nanney, D.L., 1956. Inbreeding deterioration i n Tetrahymena  pyriformis. Genetics 41: 55 (abstract). Nanney, D.L., 1957. Inbreeding degeneration i n Tetrahymena. Genetics 42: 137-146. Nanney, D.L., and P.A. Chaughey, 1955. An unstable nuclear condition i n Tetrahymena pyriformis. Genetics 40: 388-398. Nanney, D.L., and M.J. Nagel, 1964. Nuclear misbehavior i n an aberrant inbred Tetrahymena. J . Proto. 11: 465-473. Siegel, R.W., 1956. Mating types i n Oxytricha and the s i g n i f i -cance of mating types i n c i l i a t e s . B i o l . B u l l . 110: 352-357. Siegel, R.W., 1967.. Genetics of ageing and the l i f e cycle i n c i l i a t e s . In "Aspects of the Biology of Ageing". Symp. Soc. exp. B i o l . 21: 127-148. Sonneborn, T.M., 193.9. Sexuality and related problems i n Paramecium. C o l l . Net 14: 77-84. Sonneborn, T.M., 1947. Recent advances i n the genetics of Paramecium and Euplotes. Advanc;.- Genet. 1: 264-358. Sonneborn, T.M., 1957. Breeding systems, reproductive methods and species problems i n Protozoa. In "The Species 55 Problem". Amer. Assoc. Advan. Sci., Washington, D.C. pp. 155-324. Sonneborn, T.M., 1966. A non-conformist genetic system i n Paramecium aure l i a. Amer. Zool. 6: 589 (abstract). Sonneborn, T.M., and M. Schneller, 1960. Age induced muta-tions i n Paramecium. In "The Biology of Ageing". Ed. B.L. Strehler. Waverly Press Inc., Baltimore, pp. 286-287. Tartar, V.,, 1961. "The Biology of Stentor". Pergamon Press, Oxford. Taub, S., 1966. Regular changes i n mating type composition i n s e l f i n g cultures and i n mating type p o t e n t i a l i t y i n s e l f i n g caryonides of Paramecium aure l i a. Genetics 54: 173-189. Wells, C , 1965. Age associated nuclear anomalies i n Tetrahymena. J . Proto. 12: 561-563. Wichterman, R., 1953. "The Biology of Paramecium". The Blakiston Co., New York. 56 APPENDIX  Origin and History of Stocks Stock C This stock was obtained i n August 1967 from Carolina B i o l o g i c a l Supply Company (Burlington, North Carolina). It was collected i n the v i c i n i t y of Burlington and had been maintained i n culture f o r approximately 35 years. Stock DF This stock was isolated from a water sample taken from Ross Lake, B r i t i s h Columbia i n March, 1968. Stock S This stock was obtained i n June 1966 from Dr. Vance Tartar. It was collected from S t e l l a Lake, Washington and had been maintained i n culture since 1954. Stock SC This stock was obtained i n February 1968 from Schettle Bi o l o g i c a l s (P.O. Box 184, Stillwater, Minnesota). It was collected i n the v i c i n i t y of Greensboro, North Carolina and had been maintained i n culture for three years. Stock ST This stock was obtained i n February 1968.from E.G. Stein-h i l b e r + Co. (P.O. Box 888, Oshkosh, Wisconsin). It was o r i g i n a l l y started from organisms belonging to Stock SC. 57 Stock T This stock was obtained i n August 1967 from the General B i o l o g i c a l Supply House (8200 South Hoyne Ave.., Chicago, I l l i n o i s ) . It was collected i n the v i c i n i t y of Chicago and had been maintained i n continuous culture f o r approximately 15 years. Stock CH This stock was obtained i n October 1967 from Charles Harden (Science + Engineering Inc., 140 Fourth Ave., Waltham, Massachusetts). It was o r i g i n a l l y started from organisms belonging to Stock T. Stock H This stock was obtained i n November 1966 from Noel De Terra (The Institute for Cancer Research, 7701 Burlholme Ave., Philadelphia, Pennsylvania). It was o r i g i n a l l y started from organisms belonging to Stock T. Stock W This stock was obtained from Wards Natural Science Establishment (P.O. Box 1712, Rochester, New York). It had been maintained i n culture f o r three years, but i t s o r i g i n was not known. 

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