UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Commitment to autogamy in Paramecium blocks mating reactivity : implications for regulation of the sexual… Rahemtullah, Shamsa 1990

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1990_A6_7 R33.pdf [ 2.9MB ]
Metadata
JSON: 831-1.0098432.json
JSON-LD: 831-1.0098432-ld.json
RDF/XML (Pretty): 831-1.0098432-rdf.xml
RDF/JSON: 831-1.0098432-rdf.json
Turtle: 831-1.0098432-turtle.txt
N-Triples: 831-1.0098432-rdf-ntriples.txt
Original Record: 831-1.0098432-source.json
Full Text
831-1.0098432-fulltext.txt
Citation
831-1.0098432.ris

Full Text

COMMITMENT TO AUTOGAMY IN PARAMECIUM BLOCKS MATING REACTIVITY: IMPLICATIONS FOR REGULATION OF THE SEXUAL PATHWAY AND THE BREEDING SYSTEM SHAMSA RAHEMTULLAH B.Sc, University of B r i t i s h Columbia, 1985 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Zoology) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA February 1990 ^ Shamsa Rahemtullah, 1990 &f: fi In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia Vancouver, Canada Date Area \W DE-6 (2/88) ABSTRACT The c i l i a t e Paramecium t e t r a u r e l i a exhibits two major developmental pathways - the vegetative c e l l cycle and the sexual pathway. The l a t t e r manifests i t s e l f in two forms -autogamy ( s e l f - f e r t i l i z a t i o n ) and conjugation (cross-f e r t i l i z a t i o n ) between c e l l s of complementary mating types. In the normal l i f e history young c e l l s are immature for autogamy, but enter conjugation r e a d i l y . Autogamy f i r s t occurs normally at about 20 f i s s i o n s of age and conjugation disappears by 25. This study documents and analyzes the two major phenomena underlying t h i s unusual l i f e history. It shows how their interaction produces the observed pattern of immaturity, maturity, and senescence (Fig. 1). There are two p r i n c i p a l findings of t h i s study. F i r s t that commitment to autogamy leads to rapid loss of mating r e a c t i v i t y and second, that there are d i f f e r e n t starvation thresholds for i n i t i a t i o n of mating r e a c t i v i t y and autogamy. The starvation threshold for i n i t i a t i o n of mating r e a c t i v i t y is constant, while that for i n i t i a t i o n of autogamy decreases progressively as clonal age increases. During the immature period for autogamy the lag between onset of mating r e a c t i v i t y and induction of autogamy decreases with increasing clonal age. The progressive decrease in the starvation threshold required for induction of autogamy brings about the end of the mature period for conjugation. As autogamy i s i n i t i a t e d at progressively e a r l i e r stages in the growth of a culture, fewer and fewer c e l l s reach the l e v e l of starvation required for i n i t i a t i o n of mating r e a c t i v i t y prior to induction of autogamy. When the threshold for induction of autogamy becomes so low that no c e l l s develop mating r e a c t i v i t y prior to entering autogamy, the period of maturity for conjugation is over and autogamy becomes the sole sexual process for the remainder of the l i f e history. - i i i -TABLE OF CONTENTS ABSTRACT i i LIST OF FIGURES v i ACKNOWLEDGEMENTS v i i i INTRODUCTION 1 MATERIALS AND METHODS 7 Stocks and c u l t u r e s of Paramecium 7 Estab l i s h m e n t of I s o l a t i o n l i n e s and Induction of autogamy 7 P r e p a r a t i o n of t e s t e r c u l t u r e s 8 Determination of c e l l d e n s i t i e s 9 Mating r e a c t i v i t y t e s t s 9 Determination of c e l l c y c l e d u r a t i o n 11 P r e p a r a t i o n of immature and mature mass c u l t u r e s ( f o r mass c u l t u r e experiments) 12 Determination of f r a c t i o n of c e l l s committed to d i v i s i o n 13 Determination of f r a c t i o n of c e l l s i n autogamy 13 RESULTS 15 A. Mating r e a c t i v i t y t e s t s 15 B. Onset of mating r e a c t i v i t y 16 1. Asynchronous c u l t u r e s of unknown age 16 2. Mating r e a c t i v i t y onset i n D r y l ' s b u f f e r 16 3. I n i t i a t i o n of mating r e a c t i v i t y with r e s p e c t t o c e l l c y c l e stage 17 - i v -C. P e r s i s t e n c e of mating r e a c t i v i t y i n immature and mature c e l l s 17 1. N a t u r a l l y s t a r v e d , immature c e l l s 17 2. Abrupt downshift, immature c e l l s 18 3. N a t u r a l l y s t a r v e d , mature c e l l s 18 4. Abrupt downshift, mature c e l l s 19 5. Mixed mature and immature c e l l s 20 D. Loss of mating r e a c t i v i t y 21 1. Immature c e l l s ( c o n t r o l ) 22 2. Mature c e l l s 23 E. E f f e c t of c l o n a l age on s t a r v a t i o n t h r e s h o l d s f o r i n i t i a t i o n of autogamy and mating r e a c t i v i t y 24 DISCUSSION 28 R e g u l a t i o n of mating r e a c t i v i t y 28 R e g u l a t i o n of autogamy and the l i f e h i s t o r y 31 BIBLIOGRAPHY 61 -V-LIST OF FIGURES F i g u r e 1. C l o n a l l i f e h i s t o r y of Paramecium t e t r a u r e l i a . 43 F i g u r e 2. S t a b i l i z a t i o n of mating r e a c t i v i t y i n b u f f e r . 44 F i g u r e 3. Increase i n c e l l number and onset of mating r e a c t i v i t y i n a n a t u r a l l y s t a r v e d c u l t u r e of unknown age. 4 5 F i g u r e 4. Mating r e a c t i v i t y onset i n D r y l ' s b u f f e r . 46 F i g u r e 5. Mating r e a c t i v i t y onset as a f u n c t i o n of c e l l c y c l e stage occupied by c e l l s upon n u t r i e n t downshift. 47 F i g u r e 6. P e r s i s t e n c e of mating r e a c t i v i t y i n immature c e l l s . 48 F i g u r e 7. Occurrence of autogamy i n n a t u r a l l y s t a r v e d "senescent" c e l l s , mature f o r autogamy. 49 F i g u r e 8. Occurrence of autogamy and mating r e a c t i v i t y i n an a b r u p t l y s t a r v e d mature c u l t u r e . 50 F i g u r e 9. Mating r e a c t i v i t y and occurrence of autogamy i n a mixed c u l t u r e , mature and immature c e l l s . 51 F i g u r e 10. Mating r e a c t i v i t y d u r i n g the f i r s t and second c e l l c y c l e s a f t e r n u t r i e n t downshift. 52 F i g u r e 11. Percent autogamy as a f u n c t i o n of time a f t e r mating r e a c t i v i t y onset i n mature c u l t u r e s of i n c r e a s i n g c l o n a l age, 3 to 13 f i s s i o n s . 53 - v i -F i g u r e 12. Percent autogamy as a f u n c t i o n of time a f t e r mating r e a c t i v i t y onset i n mature c u l t u r e s of i n c r e a s i n g c l o n a l age, 15 to 25 f i s s i o n s . 54 F i g u r e 13. Time between i n i t i a t i o n of mating r e a c t i v i t y and i n i t i a t i o n of macronuclear fragmentation i n s t a t i o n a r y phase c u l t u r e s as a f u n c t i o n of c l o n a l age. 55 F i g u r e 14. Percent autogamy as a f u n c t i o n of c e l l d e n s i t y i n mature c u l t u r e s of i n c r e a s i n g c l o n a l age, 9 to 19 f i s s i o n s . 56 F i g u r e 15. Percent autogamy as a f u n c t i o n of c e l l d e n s i t y i n mature c u l t u r e s of i n c r e a s i n g c l o n a l age, 21 to 31 f i s s i o n s . 57 F i g u r e 16. Percent autogamy as a f u n c t i o n of c e l l d e n s i t y i n mature c u l t u r e s of i n c r e a s i n g c l o n a l age, 33 to 41 f i s s i o n s . 58 F i g u r e 17. C e l l d e n s i t y a t autogamy and i n i t i a t i o n of mating r e a c t i v i t y as a f u n c t i o n of c l o n a l age. 59 F i g u r e 18. Comparison of growth r a t e s of even and odd mating types. 60 - v i i -ACKNOWLEDGEMENTS This thesis would not have seen the l i g h t of the day but for the constant d i r e c t i o n , c r i t i q u e , and valuable guidance given me throughout the duration of t h i s study by Dr. J.D. Berger. It would be remiss of me i f I did not also g r a t e f u l l y acknowledge my appreciation to Dr. T. G r i g l i a t t i and Dr. D. Holm for the i r help in preparing t h i s t h e s i s . F i n a l l y , I owe a deep debt of gratitude to my family for t h e i r love, support and encouragement over the years. - v i i i -INTRODUCTION The two major developmental pathways in the c i l i a t e Paramecium t e t r a u r e l i a are the vegetative c e l l cycle and the sexual pathway. The sexual pathway, which includes mating, melosis, f e r t i l i z a t i o n , and macronuclear development, is entered by two d i f f e r e n t routes. One i s conjugation or r e c i p r o c a l cross-f e r t i l i z a t i o n , which is i n i t i a t e d by surface interactions between sexually reactive c e l l s of complementary mating type. The other is the i n t e r n a l l y i n i t i a t e d process of s e l f - f e r t i l i z a t i o n c a l l e d autogamy which takes place i n non- paired mature c e l l s . Since autogamy i s entered from the vegetative c e l l cycle, without the •v-.. ... a external stimulus of interacting with a reactive c e l l of complementary mating type, preparation for each c e l l cycle necessitates that the organism must determine whether the next c e l l cycle w i l l be vegetative or whether i t w i l l enter meiosis and proceed through autogamy. Both autogamy and conjugation are induced by starvation in Paramecium. When c e l l s are unable to enter either autogamy or conjugation, for example when grown continuously in the presence of excess food, the f i s s i o n rate gradually decreases and the l i n e dies out af t e r a c h a r a c t e r i s t i c number of c e l l cycles. Both autogamy and conjugation lead to f e r t i l i z a t i o n and formation of a new macronucleus, the events c r i t i c a l for i n i t i a t i o n of a new l i f e cycle and restoration of f u l l vigor. However, each process has a d i f f e r e n t pattern of occurrence in the l i f e cycle of the - 1 -organism. Age i s defined as the number of f i s s i o n s completed since the l a s t f e r t i l i z a t i o n . In general, young c e l l s r e a d i l y enter conjugation but not autogamy while older c e l l s undergo autogamy but cannot i n i t i a t e conjugation. Unlike many other c i l i a t e s which show an immature period for conjugation of s i g n i f i c a n t length (50 to 100 c e l l cycles) during which young c e l l s do not develop mating r e a c t i v i t y and thus cannot enter conjugation, P. tetzauzella shows no immature period for conjugation. That i s , c e l l s can enter conjugation even during the f i r s t c e l l cycle after f e r t i l i z a t i o n (Sonneborn, 1957, 1974). However, the period of maturity for conjugation la s t s only for 25 c e l l cycles following f e r t i l i z a t i o n . This i n t e r v a l . is about ten percent of the entire l i f e span of approximately 240 f i s s i o n s (Sonneborn, 1957, 1974). In conjugation, two c e l l s each with a macronucleus and two micronuclei meet to form a conjugant pa i r . Both micronuclei in each c e l l then undergo meiosis (two of three prezygotic d i v i s i o n s ) to produce eight haploid n u c l e i . Seven of the eight micronuclear products degenerate and the remaining micronucleus proceeds through a t h i r d prezygotic (mitotic) d i v i s i o n to y i e l d two haploid gamete nuclei or pronuclei. One of these, the migratory pronucleus of each i n d i v i d u a l , moves into the coconjugant and fuses with the stationary pronucleus to form a synkaryon. Two mitotic postzygotic d i v i s i o n s of the synkaryon then follow to y i e l d four n u c l e i , two of which become macronuclear anlagen (destined to become new macronuclei) and two -2-micronuclei. Conjugants separate during the postzygotic d i v i s i o n s . Beginning at the end of melosis, the old parental macronucleus breaks up to form about 35 rounded fragments. A d i s t i n c t i v e coarse, rope- l i k e conformation of the macronucleus c a l l e d a skein marks the early stage of t h i s process. F i n a l l y , each exconjugant containing two micronuclei and two macronuclear anlagen divides, segregating to each daughter c e l l a macronuclear anlage while the two micronuclei divide by ordinary mitosis to restore the normal nuclear condition (1 macronucleus and 2 micronuclei). Autogamy i s l i k e conjugation except that i t occurs within single unpaired c e l l s . It i s an e n t i r e l y endogenous event; external factors other than nutrient l e v e l do not play a role in invoking i t s occurrence. The re s u l t of autogamy i s that the genome of Paramecium becomes completely homozygous owing to the union of two g e n e t i c a l l y i d e n t i c a l haploid pronuclei in the same c e l l . Since the new macronucleus i s a derivative of the homozygous synkaryon or zygote nucleus, i t is also homozygous. In conjugation (cross- f e r t i l i z a t i o n ) , the synkaryon of both partners i s i d e n t i c a l , but It may be heterozygous. Commitment to autogamy i s a two- step process. The i n i t i a l commitment takes place at a point in the c e l l cycle, about 90 minutes prior to f i s s i o n , at which c e l l s become committed to d i v i s i o n (Berger, 1986). If c e l l s are subjected to nutrient downshift prior to the point of commitment to d i v i s i o n , they enter autogamy (meiosis) immediately following the next f i s s i o n . -3-I f , however, starvation is induced af t e r commitment to d i v i s i o n , the next c e l l cycle w i l l be vegetative, regardless of the nutrient l e v e l . This i n i t i a l commitment to meiosis i s con d i t i o n a l . If c e l l s are returned to growth medium, the commitment i s reversed. F i n a l commitment to meiosis occurs within 20 minutes following the pre- autogamous f i s s i o n . If c e l l s are returned to medium with excess food af t e r t h i s point, meiosis proceeds regardless. The patterns of occurrence of autogamy and conjugation d i f f e r . C e l l s are immature for autogamy during the f i r s t 15 to 20 c e l l cycles following f e r t i l i z a t i o n . In these c e l l s autogamy does not occur following b r i e f starvation as i t does in mature c e l l s . During the l a t t e r part of t h i s immature period, however, less and less starvation i s required for the c e l l s to enter autogamy (Sonneborn, 1974). The cl o n a l age at which c e l l s can no longer develop mating r e a c t i v i t y , but d i r e c t l y enter autogamy, marks the end of the period of maturity for conjugation and the beginning of senescence (Sonneborn, 1957, 1974). "Senescence" i s marked by the loss of the c a p a b i l i t y to mate. This period i s by far the longest component of the organism's l i f e h i s t o r y and may p e r s i s t for more than 200 f i s s i o n s . F i n a l l y , death of the clone marks both the end of senescence and the end of the l i f e cycle (Sonneborn, 1957, 1974). This thesis examines the control of the sexual pathway of Paramecium in order to ascertain the nature of the relationship between autogamy and conjugation. The question examined here i s -4-whether there exists a hierarchy with respect to autogamy and conjugation in mature c e l l s . In other words, i f mature c e l l s are permitted to become mating reactive before commitment to autogamy, does conjugation s t i l l proceed or does autogamy block conjugation? And, conversely, does mating r e a c t i v i t y ever develop once mature c e l l s have become committed to autogamy? The preference of one sexual pathway over the other in P. t e t r a u r e l i a has been suggested in e a r l i e r studies. Beisson and Capdeville (1966) have shown that abrupt starvation f i r s t induces mating r e a c t i v i t y and then autogamy in c e l l s that are old enough to enter either pathway at approximately the same time given suitable conditions. Mature c e l l s become mating reactive about three hours aft e r being washed in exhausted medium. They then r a p i d l y lose their mating r e a c t i v i t y as autogamy sets i n . The addition of protein synthesis i n h i b i t o r s appears to block autogamy by preventing the synthesis of s p e c i f i c proteins needed for the nuclear reorganization process. Mating reactive mature c e l l s continue to remain reactive in the presence of such i n h i b i t o r s (Beisson and Capdeville, 1966). This study shows that mating reactive mature c e l l s destined to undergo autogamy loose t h e i r r e a c t i v i t y at some point between, commitment to d i v i s i o n and f i s s i o n , which occurs 90 minutes l a t e r . One of the aims of t h i s thesis i s to determine p r e c i s e l y where shut off of mating r e a c t i v i t y occurs within t h i s 90 minute = i n t e r v a l . More importantly, t h i s thesis examines the h i e r a r c h i c a l regulation of the sexual pathway in P. t e t r a u r e l i a , -5-i . e . , how the relat i o n s h i p between autogamy and conjugation changes as f i s s i o n age increases and c e l l s pass from maturity to senescence, leaving autogamy as the sole sexual process in the l a t t e r portion of the organism's l i f e history. This information enhances our knowledge of the ecology of P. tetraurelia, i t s breeding strategy, and i t s r e l a t i o n s h i p with other members of the P. aurelia species complex and other members of the genus. - 6 -MATERIALS AND METHODS Stocks and c u l t u r e s o f Paramecium Paramecium tetraurelia Sonneborn 1975 w i l d type s tock 51-S and s t o c k d4-1001 c a r r y i n g the c c l muta t ion ( P e t e r s o n and B e r g e r , 1976; Rasmussen and B e r g e r , 1984) were grown i n phosphate -b u f f e r e d C e r o p h y l medium (Sonneborn, 1970) w i t h Klebsiella pneumoniae as the food o r g a n i s m . S tandard methods used were those of Sonneborn (1970) . c u l t u r e s were m a i n t a i n e d a t 27 C . Establishment of isolation lines and induction of autogamy S e v e r a l c e l l s were t r a n s f e r r e d from a s tock c u l t u r e of one mat ing type t o a d e p r e s s i o n s l i d e w e l l c o n t a i n i n g C e r o p h y l medium. They were then washed by b e i n g t r a n s f e r r e d , one by one from one w e l l t o the o t h e r , through f i v e more w e l l s c o n t a i n i n g medium. The c e l l s were l e f t i n the l a s t w e l l f or one hour a f t e r which t h r e e c e l l s were i n d i v i d u a l l y t r a n s f e r r e d by m i c r o p i p e t t e through t h r e e a d d i t i o n a l w e l l s c o n t a i n i n g medium and were assembled i n the l a s t w e l l . F i n a l l y , each of the t h r e e c e l l s was t r a n s f e r r e d to a s e p a r a t e w e l l w i t h medium i n a s i n g l e d e p r e s s i o n s l i d e . D a i l y I s o l a t i o n l i n e s of each mat ing type were m a i n t a i n e d s e p a r a t e l y by t r a n s f e r r i n g a c e l l from the p r e v i o u s d a y ' s w e l l to a new w e l l c o n t a i n i n g f r e s h medium. C e l l s were washed e v e r y two weeks. Autogamy was induced by s t a r v a t i o n (Sonneborn, 1950) . T h i r d - d a y - l e f t o v e r d e p r e s s i o n w e l l c u l t u r e s t h a t had reached - 7 -stationary phase were used as sources of autogamous c e l l s . The c e l l s were tested for the presence of autogamy with Dippell's s t a i n (Sonneborn, 1970). A v e i l was judged to be autogamous i f more than 90% of a sample of 30 stained c e l l s exhibited macronuclear fragmentation. Preparation of tester cultures Lines were expanded by tra n s f e r r i n g a three day old depression s l i d e well culture to a tube with 10 ml. of fresh medium. The next day, the single tube culture was di s t r i b u t e d equally among four fresh tubes and refed with 15 ml. medium to achieve a high number of c e l l s with maximum growth rate. On the t h i r d day, the top 2-3 cm. of culture from each tube were transferred to a fresh tube. Each of the four new tubes was then fed approximately 20, 15, 10, and 5 ml. of food to produce d i l u t i o n s of approximately 1:4, 1:3, 1:2, and 1:1 respectively of the o r i g i n a l culture with fresh medium. 24 hours l a t e r , c e l l s of each mating type from tube cultures having similar amounts of food were mixed together and tested for mating r e a c t i v i t y (see below). A l t e r n a t i v e l y , several flask cultures (see page 12) of each mating type were d i l u t e d with fresh medium to obtain a range of densities (500-2000 cells/ml.) by the next morning. Cultures of each mating type were separately f i l t e r e d and washed twice in Dryl's buffer when they reached a minimum density of 2000 c e l l s / m l . Approximately 3.5 hours after washing in Dryl's buffer the cultures became mating reactive and were used as a source of - 8 -reactive testers as long as high mating r e a c t i v i t y was present (8-16 h). A v a i l a b i l i t y of testers that were 40-50% mating reactive throughout the day was ensured by se t t i n g up a d i l u t i o n series on the previous day. Determination of cell densities Population density was estimated by manually counting c e l l s . Samples of 0.05 ml. were removed with an Eppendorf pipetter from a thoroughly s t i r r e d culture. The c e l l s were ejected into 1 ml. of medium in a depression s l i d e well. Formalin (0.05 ml.) was added to k i l l the c e l l s , and the dead c e l l s were then counted as they were withdrawn with a micropipette. Each count was the mean of three samples (at least 100 cells/sample). The count was multiplied by the appropriate d i l u t i o n factor to give the number of c e l l s / m l . As a standard medium was used throughout these experiments, c e l l density was used as a measure of the degree of starvation and the a v a i l a b i l i t y of food. Mating r e a c t i v i t y tests Approximately 0.5 ml. of culture of each mating type was placed in the middle well of a depression s l i d e . The same volume of each was added separately to each of the two end wells. These cultures served as controls for possible contamination of the tester cultures. Mating r e a c t i v i t y was scored. Three procedures were used in various experiments to estimate the degree of mating - 9 -r e a c t i v i t y . (1) Mating r e a c t i v i t y was s c o r e d 10 minutes a f t e r the t e s t e r s t r a i n s were mixed t o g e t h e r . The degree of mating r e a c t i v i t y was estimated u s i n g a r a n k i n g s c a l e r a nging from 0 to 5. Zero corresponded to no observable mating r e a c t i v i t y (absence of p a i r s or clumps) while f i v e corresponded to the h i g h e s t degree of mating r e a c t i v i t y observed i n e q u i v a l e n t mixtures of t e s t e r s t r a i n s . (2) As the a r b i t r a r y s c a l e d e s c r i b e d above gave o n l y a r e l a t i v e measure of mating r e a c t i v i t y , a s i n g l e c e l l mating r e a c t i v i t y t e s t was designed to measure the percentage of mating r e a c t i v i t y . A drop of D r y l ' s b u f f e r was p l a c e d i n each of 42 d e p r e s s i o n s l i d e w e l l s . A s i n g l e experimental c e l l was p l a c e d i n each d e p r e s s i o n w e l l . An excess of r e a c t i v e t e s t e r s of complementary mating type prepared i n D r y l ' s b u f f e r was added t o each w e l l . A p o s i t i v e mating r e a c t i o n was noted i f a p a i r or clump was observed one hour a f t e r t e s t e r s were added. The time of the t e s t was taken to be the time a t which the t e s t e r s were added to the experimental c e l l s . The percentage mating r e a c t i v i t y was taken as the f r a c t i o n of the 42 experimental c e l l s mating. (3) A known number of experimental c e l l s (30-42) was p l a c e d i n 0.5 ml. D r y l ' s b u f f e r i n a d e p r e s s i o n w e l l . An excess of r e a c t i v e t e s t e r s of complementary mating type was added to the w e l l . The number of p a i r s observed one hour a f t e r the t e s t e r s were added was taken to be the f r a c t i o n of -10-experimental c e l l s added that were mating react ive at the time of addi t ion of t e s t e r s . Determination of cell cycle durat ion a. Control cells The c e l l cycle durat ion of contro l c e l l s was determined by t r a n s f e r r i n g approximately 25 d i v i d e r s from a P e t r i plate cu l ture (see page 12) in normal medium to a s ingle depression well containing normal medium within a b r i e f (2- 3 min.) i n t e r v a l . Four to f ive hours a f ter c o l l e c t i o n of the d i v i d e r s , a s ingle c e l l was placed in each of 42 wells containing normal medium. The wells were then checked p e r i o d i c a l l y (every 20 min.) for the presence of d i v i d e r s or d iv ided c e l l s s t a r t i n g about 5.5 hours a f ter f i s s i o n . The cumulative number of c e l l s having reached d i v i s i o n was recorded at each i n t e r v a l u n t i l more than 75% of the c e l l s had d i v i d e d . The number of d iv ided c e l l s was then plot ted as a function of time. The median time of d i v i s i o n was estimated by ex trapo la t ion . b. Cells subjected to nutr ient downshift Approximately 25 d iv iders were transferred from a P e t r i plate cu l ture in normal medium to a depression well containing normal medium. Some time la t er (2-4 h) , the d i v i d e r s were washed through three wells containing D r y l ' s buf fer . Single c e l l s were placed In wells containing D r y l ' s so lu t ion four to f ive hours -11-a f t e r nutrient downshift. The median time of d i v i s i o n was determined as described above. Preparation of immature and mature mass .cultures (for mass culture experiments) a. Production of mature cultures C e l l s were washed and maintained in i s o l a t i o n l i n e cultures. Two days l a t e r , a plate (14 depression s l i d e s , 42 v e i l s ) of single c e l l Isolation cultures was made from a single healthy v e i l . The i n i t i a l s l i d e vas saved as an autogamy control ( i . e . to determine whether the parent c e l l s were mature as indicated by autogamy in a high percentage of the control c u l t u r e ) . Mature c e l l s undergo autogamy when food i s exhausted. The next day c e l l s from s i x well cultures were transferred to a separate P e t r i plate. At least one depression s l i d e well culture was saved as an autogamy control - i F i n a l l y , on the next day, the P e t r i plate cultures were pooled in a flask and d i l u t e d as desired to obtain mature cultures of various d e n s i t i e s . b. Production of immature cultures To obtain an immature culture, a mature culture in which more than 95% of c e l l s were in autogamy was d i l u t e d with fresh medium. -12-Determination of fraction of cells committed to division Paramecium c e l l s become committed to d i v i s i o n about 90 minutes prior to f i s s i o n (Ching and Berger, 1986; Berger and Ching, 1988). This i s r e a d i l y observed in c e l l s carrying the ccl mutation which re s u l t s in rapid c e l l cycle arrest following transfer of the c e l l s to the r e s t r i c t i v e temperature (34.4 C). However, i f c e l l s have already reached the point of commitment to d i v i s i o n , they continue to f i s s i o n a f t e r transfer to r e s t r i c t i v e conditions. Synchronous samples of 42 c c l c e l l s were placed in 0.5 ml. of fresh medium in 1.5 ml. tubes and placed in a waterbath at 34.4 C. One hour aft e r control c e l l s had completed f i s s i o n , the f r a c t i o n of c e l l s d i v i d i n g under r e s t r i c t i v e conditions was determined. This value was used as an estimator of the f r a c t i o n of c e l l s committed to d i v i s i o n at the time of transfer to r e s t r i c t i v e conditions. Determination of fraction of cells in autogamy To determine the f r a c t i o n of c e l l s completing meiosis, 100 experimental c e l l s were k i l l e d by drying on albumen- coated s l i d e s at each time of c o l l e c t i o n . The s l i d e s were stained with Azure A and the number of c e l l s in each sample that had reached the stage of macronuclear fragmentation in autogamy (skein stage or la t e r ) was scored. The skein stage, a thick, coarse rope-l i k e conformation of the macronucleus i s a convenient marker because i t i s e a s i l y i d e n t i f i a b l e in stained c e l l s at low magnification. It appears just after completion of meiosis, at -13-4.5 hours aft e r the pre- autogamous f i s s i o n (Berger, 1986). -14-RESULTS A. Mating r e a c t i v i t y tests The mating reaction i s the f i r s t step in the conjugation pathway in Paramecium. Consequently, assessment of mating r e a c t i v i t y i s a key requirement for analysis of i n i t i a t i o n of the sexual pathway in conjugants. Three procedures were developed to estimate the degree of mating r e a c t i v i t y i n experimental c e l l s (Methods, page 9). Of these, measurement of mating r e a c t i v i t y using a ranking scale was the most subjective. This method i s quick and easy, but not quantitative. There i s no assurance that the spacing between levels in the scale i s uniform. Consequently, quantitative single c e l l and multiple c e l l mating r e a c t i v i t y tests were also developed (Methods, page 9). These tests gave quantitative, repeatable estimates of the f r a c t i o n of the population entering conjugation. Wherever possible, the l a t t e r two procedures were employed. The optimum timing for scoring mating r e a c t i v i t y tests was examined. Single c e l l mating r e a c t i v i t y tests (Methods, page 9) were set and the percentage mating r e a c t i v i t y was scored at f i v e minute intervals (Fig. 2). The percentage of c e l l s finding mates reached a stable maximum value by 30 to 45 minutes after testers were added. Therefore, subsequent mating r e a c t i v i t y tests were scored 60 minutes aft e r addition of testers to provide a margin of assurance. -15-8. Onset of mating r e a c t i v i t y 1. Asynchronous cultures of unknown age The i n i t i a l task was to est a b l i s h the conditions necessary for inducing mating r e a c t i v i t y . 5 ml. of culture was d i l u t e d with various amounts of fresh medium (Methods, page 8). 24 to 30 hours after feeding, the 1:1 tube showed strong mating r e a c t i v i t y and the 1:2 tube a much lower l e v e l . This provided a source of mating reactive c e l l s . Since nutrient l i m i t a t i o n plays a major role in inducing mating r e a c t i v i t y , c e l l density and mating r e a c t i v i t y were examined as a culture entered stationary phase. The i n i t i a l c e l l density was about 2800 c e l l s / m l . The rate of population growth declined over a period of four to s i x hours (Fig. 3). Mating r e a c t i v i t y f i r s t appeared at a density of 3400 c e l l s / m l . , but i t did not gain strength u n t i l several hours after the growth rate declined to low l e v e l s . By t h i s time the c e l l , density had s t a b i l i z e d at 4000 c e l l s / m l . 2. Mating r e a c t i v i t y onset in Dryl's buffer To determine the time of onset of mating r e a c t i v i t y i n c e l l s washed into Dryl's buffer, exponential growth phase cultures of complementary mating type (density 2000 cells/ml.) were washed twice by gentle centrifugation in Dryl's buffer, resuspended in buffer and tested at various times for mating r e a c t i v i t y using the ranking scale method (Methods, page 8). Mating r e a c t i v i t y c o n sistently appeared between 3.25 and 3.50 hours following -16-washing and persisted for at least three hours (Fig. 4). This observation made i t possible to r e l i a b l y obtain reactive testers ..from mass cultures. 3. I n i t i a t i o n of mating reactivity vith respect to cell cycle stage I n i t i a t i o n of mating r e a c t i v i t y i s not c e l l cycle stage dependent. The 3.3 hr in t e r v a l observed between the time that a culture was washed into Dryl's buffer and the onset of mating r e a c t i v i t y did not change with c e l l cycle stage when sets of synchronous c e l l s were washed into Dryl's buffer at d i f f e r e n t positions within the c e l l cycle (Fig. 5). C. Persistence of mating reactivity in immature and mature cells The persistence of mating r e a c t i v i t y after i t s i n i t i a l onset was then examined. I f i r s t examined the pattern of mating r e a c t i v i t y in immature c e l l s which are unable to undergo autogamy. This was important owing to possible interference of autogamy with the pattern of mating r e a c t i v i t y . 1. Naturally starved, immature c e l l s C e l l density and the percentage of mating reactive c e l l s in a na t u r a l l y starved immature culture were scored over a period of 20 hours (Fig. 6). In t h i s experiment, mating r e a c t i v i t y f i r s t appeared when c e l l density was about half of i t s f i n a l l e v e l (2300 c e l l s / m l . ) , increased r a p i d l y for the next s i x hours and -17-then decreased s l o w l y , p e r s i s t i n g a t low l e v e l s throughout the remainder of the 20 hour o b s e r v a t i o n p e r i o d . Thus, there i s extremely long p e r s i s t e n c e of mating r e a c t i v i t y i n a t l e a s t some c e l l s i n the c u l t u r e . 2. Abrupt downshift, immature cells S i m i l a r r e s u l t s were obtained when c e l l s were s u b j e c t e d to abrupt n u t r i e n t downshift by being washed i n t o D r y l ' s b u f f e r ( F i g . 6 ) . The main d i f f e r e n c e between t h i s experiment and t h a t with n a t u r a l l y s t a r v e d c e l l s was the s l i g h t l y more a c c e l e r a t e d l o s s of mating r e a c t i v i t y such t h a t mating r e a c t i v i t y disappeared by 18 hours a f t e r washing. 3. Naturally starved, mature cells The c e l l d e n s i t y , percentage of c e l l s completing meiosis (Methods, page 13), and percentage mating r e a c t i v i t y of a mature c u l t u r e was f o l l o w e d over time ( F i g . 7). The f r a c t i o n of c e l l s completing m e i o s i s f o l l o w e d the p a t t e r n of i n c r e a s e i n c e l l number. Throughout t h i s p e r i o d , no mating r e a c t i v i t y was observed. The preautogamous f i s s i o n takes p l a c e 4.5 hours p r i o r t o macronuclear s k e i n formation (Berger, 1986). A preautogamous f i s s i o n curve can thus be c o n s t r u c t e d by s h i f t i n g the curve showing macronuclear fragmentation back 4.5 hours. Since c e l l s t r a v e r s e the f i n a l p o i n t of commitment to autogamy w i t h i n minutes f o l l o w i n g the preautogamous f i s s i o n , the preautogamous f i s s i o n -18-curve can be taken as a good i n d i c a t o r of the k i n e t i c s with which c e l l s become committed to autogamy. The curve shows t h a t the c e l l d e n s i t y a t which mature c e l l s become committed to autogamy i s f a r lower than t h a t a t which mating r e a c t i v i t y u s u a l l y appears (about 1000 c e l l s / m l . vs. 3500 c e l l s / m l . ) . The f o r e g o i n g suggests t h a t two d i f f e r e n t s t a r v a t i o n t h r e s h o l d s e x i s t , one f o r autogamy and one f o r mating r e a c t i v i t y . As a mature c u l t u r e e n t e r s s t a t i o n a r y phase, autogamy appears to occur w e l l before mating r e a c t i v i t y c o u l d develop. In other words, c e l l s do not have t o be n e a r l y as s t a r v e d to become committed to autogamy as they do to develop mating r e a c t i v i t y . The r e s u l t s a l s o i n d i c a t e t h a t e n t r y i n t o autogamy b l o c k s development of mating r e a c t i v i t y . 4. Abrupt downshift, mature cells A mature c u l t u r e with an approximate s t a r t i n g c e l l d e n s i t y of 2000 c e l l s / m l . was washed i n D r y l ' s b u f f e r . As i n p r e v i o u s experiments, mating r e a c t i v i t y appeared approximately 3.5 hours a f t e r downshift ( P i g . 8). T h e r e a f t e r i t decreased r a p i d l y and disappeared by nine hours f o l l o w i n g downshift. In F i g u r e 8 mating r e a c t i v i t y i s shown as the percentage of c o n t r o l r e a c t i v i t y (45%). In most of the experiments, the c o n t r o l s had a percent mating r e a c t i v e score of approximately 40 p e r c e n t . In a l l cases the c o n t r o l value was taken to be 100 percent. The r a p i d l o s s of mating r e a c t i v i t y was a l r e a d y underway as c e l l s completed the preautogamous f i s s i o n . Moreover, the i n i t i a l -19-mating r e a c t i v i t y was 80% of the contro l value which corresponds to the estimated 20% of c e l l s that had completed the preautogamous f i s s i o n at th i s po int . This implies that react ive mature c e l l s loose c a p a b i l i t y to remain mating react ive some time before the preautogamous f i s s i o n , presumably soon af ter they traverse the i n i t i a l point of commitment to autogamy. Therefore, as c e l l s become committed to autogamy t h e i r entry into the conjugation pathway seems to be blocked. 5. Naturally starved, mature and Immature cells, mixed If d i f f e r e n t s tarvat ion thresholds for autogamy and conjugation do indeed e x i s t , then a n a t u r a l l y starved mixed cu l ture should show onset of autogamy in mature c e l l s well before mating r e a c t i v i t y develops in immature c e l l s . Mature and immature c e l l s were grown separately in P e t r i p l a t e s . On the day of the experiment, two plates containing immature c e l l s and three containing mature c e l l s (about 1000 c e l l s / m l . ) were combined in a f l a s k . This c e l l dens i ty was chosen to ensure that mature c e l l s had not yet begun meiosis ( F i g . 9) . The c e l l dens i ty , percentage completing meiosis , and percentage of mating react ive c e l l s were fol lowed. The median point of onset of mating r e a c t i v i t y occurred at a c e l l densi ty of 3500 c e l l s / m l . ( F i g . 9) . I f i n i t i a t i o n of mating r e a c t i v i t y was tr iggered 3.5 hours e a r l i e r , as indicated by the abrupt downshift experiments, the c e l l densi ty at the point of i n i t i a t i o n of mating r e a c t i v i t y would be 2500 c e l l s / ml. At th i s -20-p o i n t (3.6 h ) , more than a t h i r d of the mature c e l l s had reached the stage of macronuclear fragmentation. I n i t i a l commitment to autogamy occurs 6 hours p r i o r to macronuclear fragmentation and would have taken p l a c e a t a c e l l d e n s i t y below 1000 c e l l s / m l . That i n i t i a l commitment to autogamy occurs s e v e r a l hours before mating r e a c t i v i t y i s t r i g g e r e d and occurs a t a much lower c e l l d e n s i t y i n d i c a t e s t h a t d i f f e r e n t s t a r v a t i o n t h r e s h o l d s e x i s t f o r autogamy and c o n j u g a t i o n ( l e s s than 1000 c e l l s / m l . and about 3500 c e l l s / ml., r e s p e c t i v e l y ) . Commitment to autogamy a l s o appears to o v e r r i d e entrance i n t o the c o n j u g a t i o n pathway i n mature c e l l s . D. Loss of mating reactivity The pr e c e d i n g experiments s t r o n g l y suggest t h a t autogamous c e l l s are not mating r e a c t i v e . They not o n l y c o n f i r m e a r l i e r o b s e r v a t i o n s t h a t commitment to autogamy o v e r r i d e s entrance i n t o the c o n j u g a t i o n pathway i n mature c e l l s , but they a l s o suggest t h a t mating r e a c t i v e mature c e l l s , having t r a v e r s e d the i n i t i a l p o i n t of commitment to autogamy, must l o s e s e x u a l r e a c t i v i t y p r i o r to the preautogamous f i s s i o n s i n c e f i n a l commitment to autogamy occurs w i t h i n 20 minutes a f t e r the preautogamous f i s s i o n (Berger, 1986). Loss of mating r e a c t i v i t y was determined by measuring mating r e a c t i v i t y d u r i n g the l a t t e r p a r t of the f i r s t c e l l c y c l e a f t e r n u t r i e n t downshift. When n u t r i e n t downshift occurs p r i o r t o the p o i n t of commitment to d i v i s i o n m eiosis begins soon a f t e r the f o l l o w i n g f i s s i o n (Berger, 1986). I -21-t h e r e f o r e examined the p a t t e r n of mating r e a c t i v i t y i n synchronous c e l l samples d u r i n g both the f i r s t c e l l c y c l e ( i n which the n u t r i e n t downshift occurred) and i n the second c e l l c y c l e (during which autogamy occurs) i n mature c e l l s s u b j e c t e d to abrupt n u t r i e n t downshift. 1. Immature cells (control) Immature c e l l s do not undergo autogamy. T h e r e f o r e , c e l l s t h a t develop mating r e a c t i v i t y i n the f i r s t c e l l c y c l e should r e t a i n r e a c t i v i t y through a t l e a s t a p o r t i o n of the second c e l l c y c l e . To demonstrate t h i s p o i n t d i v i d e r s were c o l l e c t e d from immature P e t r i p l a t e c u l t u r e s and washed i n D r y l ' s b u f f e r 2 hours a f t e r f i s s i o n , e a r l y enough so t h a t they c o u l d become mating r e a c t i v e w e l l before f i s s i o n . Mating r e a c t i v i t y appeared about 18 hours before f i s s i o n and continued s t e a d i l y u n t i l a t l e a s t 50 percent of the c e l l s had d i v i d e d and had entered the next c e l l c y c l e ( F i g . 10). T h e r e f o r e , mating r e a c t i v i t y does not appear to be blocked i n immature c e l l s completing the f i r s t c e l l c y c l e . Repeat experiments gave s i m i l a r r e s u l t s . Mating r e a c t i v i t y i n immature c e l l s proceeds without i n t e r r u p t i o n from the time of i t s onset i n the f i r s t c e l l c y c l e through to a t l e a s t s e v e r a l hours f o l l o w i n g the f i r s t f i s s i o n a f t e r downshift. To examine the p e r s i s t e n c e of mating r e a c t i v i t y i n immature c e l l s d u r i n g the second c e l l c y c l e a f t e r downshift, d i v i d e r s were c o l l e c t e d from P e t r i p l a t e c u l t u r e s of immature c e l l s and washed i n D r y l ' s b u f f e r approximately three hours l a t e r to a l l o w onset -22-of mating r e a c t i v i t y before f i s s i o n . Sets of synchronous d i v i d e r s were c o l l e c t e d a t the next f i s s i o n . Mating r e a c t i v i t y t e s t s were then c a r r i e d out on the s e t s of newly synchronized c e l l s . Mating r e a c t i v i t y was maintained a t approximately the same l e v e l as i n the f i r s t c e l l c y c l e f o r about two hours and then g r a d u a l l y d e c l i n e d d u r i n g the next f i v e hours ( F i g . 10). Mating r e a c t i v i t y p e r s i s t e d a t low l e v e l s a f t e r t h i s f i v e hour p e r i o d . 2. Mature cells I f autogamy b l o c k s c o n j u g a t i o n , then mature c e l l s t h a t develop mating r e a c t i v i t y i n the f i r s t c e l l c y c l e a f t e r n u t r i e n t downshift and subsequently become committed to autogamy should show l o s s of mating r e a c t i v i t y . To t h i s end, the p a t t e r n of mating r e a c t i v i t y was examined i n both the f i r s t and second c e l l c y c l e s of mature c e l l s (30 to 50 f i s s i o n s o l d ) . Using the same procedure t h a t was employed f o r the immature c e l l experiments, mature c e l l s were c o l l e c t e d and washed. They become mating r e a c t i v e s l i g h t l y more than three hours before the mean time of the preautogamous f i s s i o n ( F i g . 10). The f i r s t mating r e a c t i v i t y t e s t was c a r r i e d out three hours before f i s s i o n a t which time 40% of the c e l l s were r e a c t i v e . T h i s corresponds to the c o n t r o l value (40%). Mating r e a c t i v i t y then d e c l i n e d r a p i d l y . No mating r e a c t i v i t y was present 30 minutes p r i o r to the preautogamous f i s s i o n i n any experiment and complete l o s s by one hour p r i o r to f i s s i o n o ccurred In one of three experiments. -23-As mature c e l l s t r a v e r s e the i n i t i a l p o i n t of commitment to autogamy they l o s e mating r e a c t i v i t y d u r i n g the f o l l o w i n g 30 to 60 minutes. Mature c e l l s t h a t have become committed to autogamy and l o s e mating r e a c t i v i t y by the end of the f i r s t c e l l c y c l e remain non-r e a c t i v e d u r i n g the second c e l l c y c l e while undergoing autogamy ( F i g . 10). In t h i s experiment mature c e l l s , which developed mating r e a c t i v i t y i n the f i r s t c e l l c y c l e , were r e s y n c h r o n i z e d a t f i s s i o n . . M a t i n g r e a c t i v i t y t e s t s were then c a r r i e d out a t 15 minute i n t e r v a l s f o r s e v e r a l hours d u r i n g the second c e l l c y c l e a f t e r downshift. There was no mating r e a c t i v i t y through the e n t i r e o b s e r v a t i o n a l p e r i o d . I conclude t h a t i n i t i a t i o n of autogamy b l o c k s the development of mating r e a c t i v i t y . E. E f f e c t of clonal age on s t a r v a t i o n thresholds for initiation of autogamy and mating r e a c t i v i t y The preceding experiments show t h a t i n i t i a t i o n of autogamy bl o c k s mating r e a c t i v i t y and t h a t d i f f e r e n t s t a r v a t i o n t h r e s h o l d s e x i s t f o r c o n j u g a t i o n and autogamy i n mature c e l l s . The o b s e r v a t i o n t h a t autogamy does not occur r e a d i l y i n young c e l l s but i s i n i t i a t e d r e a d i l y a t low s t a r v a t i o n l e v e l s i n mature c u l t u r e s i m p l i e s t h a t the s t a r v a t i o n t h r e s h o l d r e q u i r e d f o r the i n i t i a t i o n of autogamy decreases as c e l l s mature. I t h e r e f o r e examined the a s s o c i a t i o n between s t a r v a t i o n l e v e l s , onset of mating r e a c t i v i t y , and completion of autogamy i n c u l t u r e s of d i f f e r e n t c l o n a l age. C u l t u r e s of known c l o n a l age were prepared - 2 4 -by s e q u e n t i a l d i l u t i o n of an autogamous c u l t u r e . I n i t i a l l y , four d i l u t i o n s were made and grown i n separate f l a s k s (1:1, 1:4, 1:16, and 1:64) to g i v e c u l t u r e s t h a t were one, t h r e e , f i v e , and seven f i s s i o n s o l d , r e s p e c t i v e l y , on r e a c h i n g f u l l s t a t i o n a r y phase d e n s i t y . A f o u r - f o l d d i l u t i o n of the 1:64 c u l t u r e was used to o b t a i n a c u l t u r e t h a t would reach an age of nine f i s s i o n s on e n t e r i n g s t a t i o n a r y phase. Subsequent f o u r - f o l d d i l u t i o n s were made from the immediately preceding c u l t u r e i n the regimen when i t had reached a d e n s i t y of about 500 c e l l s / m l . T h i s ensured t h a t the newest c u l t u r e had not yet become committed t o autogamy a t the time of d i l u t i o n . T h i s process was continued u n t i l a c l o n a l age of 41 c y c l e s was reached. In the f i r s t of two experiments, the time t h a t autogamy appeared a f t e r the onset of mating r e a c t i v i t y was examined. In the one- f i s s i o n o l d c u l t u r e , macronuclear fragmentation f i r s t appeared about 240 hours a f t e r the onset of mating r e a c t i v i t y . As the c e l l s became o l d e r , autogamy appeared p r o g r e s s i v e l y e a r l i e r and the r a t e a t which c e l l s entered autogamy i n c r e a s e d ( P i g . 11). At 17 f i s s i o n s autogamy was i n i t i a t e d a t the same time as mating r e a c t i v i t y ( F i g . 12). By 21 f i s s i o n s of age the f i r s t c e l l s completed autogamy as e a r l y as seven hours b e f o r e the onset of mating r e a c t i v i t y ( F i g . 12). In four f u r t h e r d i l u t i o n s (27, 29, 31 and 33 f i s s i o n s o l d ) mating r e a c t i v i t y was not observed (data not shown). As the c l o n a l age of the c e l l s i n c r e a s e d , autogamy appeared e a r l i e r and e a r l i e r u n t i l i t e v e n t u a l l y superseded development of mating r e a c t i v i t y ( F i g . 13). That the four o l d e s t c u l t u r e s entered autogamy without d e v e l o p i n g mating r e a c t i v i t y suggests the f o l l o w i n g : As c l o n a l age i n c r e a s e s , the s t a r v a t i o n t h r e s h o l d r e q u i r e d f o r the i n i t i a t i o n of autogamy decreases u n t i l a l l c e l l s become committed to autogamy before any of them reach the mating r e a c t i v i t y t h r e s h o l d . At t h i s p o i n t , no mating r e a c t i v i t y o c c u r s . Experiment 2 examined the c e l l d e n s i t y a t which c e l l s e n tered autogamy. F l a s k s were s t o r e d o v e r n i g h t a t 17 C to slow down growth d u r i n g unmonitored p e r i o d s and f a c i l i t a t e a c c u r a t e c e l l d e n s i t y measurements d u r i n g monitored p e r i o d s . T h i s d i f f e r e d from the f i r s t experiment, i n which a l l f l a s k s were kept a t room temperature throughout the procedure. In the youngest c u l t u r e s (1 through 13 f i s s i o n s o l d ) autogamy began at the f u l l s t a t i o n a r y phase c e l l d e n s i t y of 5000 to 5500 c e l l s / m l . ( F i g . 14). As the c e l l s became o l d e r , autogamy began at p r o g r e s s i v e l y lower c e l l d e n s i t i e s . , 4 F i g . 15). F i n a l l y , the o l d e s t c u l t u r e s (35 to 41 f i s s i o n s old) showed the f i r s t completion of autogamy at a d e n s i t y of 1000 c e l l s / m l . ( F i g . 16). Less and l e s s s t a r v a t i o n was r e q u i r e d f o r i n i t i a t i o n of autogamy as the m a t u r i t y of the c e l l s i n c r e a s e d . F i g u r e 17 compares the c e l l d e n s i t y a t median autogamy (where 50% of the c e l l s have completed autogamy) to the estimated c e l l d e n s i t y a t commitment to autogamy. The l a t t e r curve was c o n s t r u c t e d by u s i n g a standard growth curve to c a l c u l a t e the c e l l d e n s i t y a t the p o i n t of commitment t o autogamy which occurs s i x hours p r i o r t o macronuclear fragmentation. The youngest c u l t u r e s d i d not -26-i n i t i a t e autogamy u n t i l s t a t i o n a r y phase (5000 c e l l s / m l . ) while the o l d e s t c u l t u r e s i n i t i a t e d autogamy at a d e n s i t y of approximately 875 c e l l s / m l . The estimated c e l l d e n s i t y a t commitment to autogamy decreased i n an approximately l i n e a r f a s h i o n with i n c r e a s i n g c l o n a l age. F i g u r e 17 a l s o summarizes the r e s u l t s of Experiment 1 which shoved t h a t mating r e a c t i v i t y appeared a t a c o n s i s t e n t c e l l d e n s i t y (3000-3500 c e l l s / m l . ) i n a l l c u l t u r e s through 25 f i s s i o n s i n age, and t h a t no mating r e a c t i v i t y appeared i n o l d e r c u l t u r e s . -27-DISCUSSION The p r i n c i p a l f i n d i n g s of t h i s study are t h a t commitment to autogamy r e s u l t s i n l o s s of mating r e a c t i v i t y and t h a t the s t a r v a t i o n t h r e s h o l d r e q u i r e d f o r i n i t i a t i o n of autogamy decreases as c e l l s age, while t h a t f o r i n i t i a t i o n of mating r e a c t i v i t y does not. Together the o b s e r v a t i o n s p r o v i d e an e x p l a n a t i o n both f o r the presence of the immature p e r i o d f o r autogamy and f o r the l o s s of mating r e a c t i v i t y a t the end of the p e r i o d of m a t u r i t y f o r c o n j u g a t i o n . The d i s c u s s i o n w i l l d e a l f i r s t with r e g u l a t i o n of mating r e a c t i v i t y and then with r e g u l a t i o n of autogamy and i t s i m p l i c a t i o n s f o r the l i f e h i s t o r y and breeding s t r a t e g y of the organism. R e g u l a t i o n of mating r e a c t i v i t y Development of mating r e a c t i v i t y does not appear to be c e l l c y c l e stage dependent. T h i s hypothesis was suggested i n e a r l i e r s t u d i e s by Miwa and Umehara (1983) who demonstrated t h a t G2 phase c e l l s (4C micronuclear DNA content) can conjugate with G l phase c e l l s (2C micronuclear DNA content) i n stock h r d , P. t e t r a u r e l i a . In my experiments, the i n t e r v a l between the time t h a t a c u l t u r e was washed i n t o D r y l ' s b u f f e r and the onset of mating r e a c t i v i t y d i d not change with c e l l c y c l e s t a g e . However, the c e l l c y c l e stage of c e l l s a t onset of mating r e a c t i v i t y 3.3 hours a f t e r washing was not known. N u t r i e n t downshift s i g n i f i c a n t l y i n c r e a s e s c e l l c y c l e l e n g t h making i t d i f f i c u l t to -28-determine the c e l l c y c l e stage of c e l l s a t I n i t i a t i o n of mating r e a c t i v i t y (Rasmussen, 1967). The molecular s i g n a l s t h a t i n i t i a t e mating and t h a t lead to c o n j u g a t i o n i n Paramecium are not f u l l y understood. In c e r t a i n c i l i a t e s , such as Blepharisma, mating- type substances occur as s o l u b l e pheromones (gamones) i n the c e l l - f r e e f l u i d , and have been i s o l a t e d and c h a r a c t e r i z e d (Miyake and Beyer, 1973). In Paramecium, however, mating substances are i n t e g r a l p r o t e i n s of the c i l i a r y membrane (Metz, 1954; Hiwatashi, 1969; Watanabe, 1977; Kitamura and Hiwatashi, 1978). G e n e r a l l y b e l i e v e d t o be g l y c o p r o t e i n s (Brock, 1965; C r a n d a l l et a l . , 1974; Wiese, 1974; Kitamura and Hiwatashi, 1978), these mating substances mediate an i n i t i a l r e c o g n i t i o n event. The i n i t i a l adhesive i n t e r a c t i o n s i n i t i a t e subsequent responses which i n c l u d e m e i o s i s and f e r t i l i z a t i o n . The present experiments show t h a t l o s s of mating r e a c t i v i t y i s a d i r e c t consequence of i n i t i a t i o n of autogamy. The e n t i r e p o p u l a t i o n of c e l l s mature f o r autogamy (27 f i s s i o n s of age or o l d e r ) entered autogamy without ever d e v e l o p i n g mating r e a c t i v i t y when allowed to exhaust t h e i r food supply. However, when these mature c e l l s were s u b j e c t e d to abrupt downshift, mating r e a c t i v i t y developed i n most c e l l s but disappeared r a p i d l y t h e r e a f t e r . A n a l y s i s of the k i n e t i c s of i n i t i a t i o n of autogamy i n these c e l l s shows t h a t the r a p i d l o s s of mating r e a c t i v i t y was a s s o c i a t e d with commitment to autogamy. S i m i l a r o b s e r v a t i o n s have been r e p o r t e d by Beisson and C a p d e v i l l e (1966). -29-Beisson and C a p d e v i l l e noted t h a t when the p r o t e i n s y n t h e s i s i n h i b i t o r puromycin was added to mature c e l l s a t the end of the washing p r o c e s s , those c e l l s becoming mating r e a c t i v e remained mating r e a c t i v e f o r up to two weeks while autogamy was completely i n h i b i t e d . When puromycin was removed and c e l l s were resuspended i n exhausted medium, mating r e a c t i v i t y disappeared and autogamy began. T h i s suggests t h a t autogamy r e q u i r e s the s y n t h e s i s of new p r o t e i n s which t r i g g e r the l o s s of and subsequent replacement of f l a g e l l a r membrane c o n t a i n i n g the mating substances. A d d i t i o n of puromycin a t or a f t e r the second hour d i d not stop the process of autogamy (Beisson and C a p d e v i l l e , 1966). T h i s t r a n s i t i o n p o i n t probably corresponds to the p o i n t of f i n a l commitment to autogamy which occurs minutes a f t e r the preautogamous f i s s i o n and about 110 minutes a f t e r the p o i n t of i n i t i a l commitment to autogamy (Berger, 1986). My mating r e a c t i v i t y c o n t r o l v a l u e s are not as high as those obtained by Beisson and C a p d e v i l l e (1966) probably because of d i f f e r e n c e s i n washing procedure. Beisson and C a p d e v i l l e washed t h e i r t e s t e r s i n exhausted medium and obtained 90% mating r e a c t i v i t y . On the other hand, I washed my t e s t e r s i n D r y l ' s b u f f e r and achieved lower mating r e a c t i v i t y l e v e l s — t y p i c a l l y 40% t o 50%. T h i s d i f f e r e n c e c o u l d be due to the d e l e t e r i o u s e f f e c t s of the non- n u t r i e n t b u f f e r . The presence of mature c e l l s (30%) i n the t e s t e r mixture c o u l d a l s o account f o r the low l e v e l s of mating r e a c t i v i t y o b t a i n e d . -30-R e g u l a t i o n of autogamy and the l i f e h i s t o r y The two forms of the s e x u a l pathway i n P. t e t r a u r e l i a , c o n j u g a t i o n and autogamy, occur d u r i n g d i f f e r e n t phases of the organism's l i f e h i s t o r y . T h i s study shows t h a t the occurrence of mating r e a c t i v i t y i s r e s t r i c t e d to the f i r s t 10% of the c l o n a l l i f e span and autogamy i s v i r t u a l l y the s o l e sexual process d u r i n g the remainder of the l i f e h i s t o r y . The t i m i n g of i n i t i a t i o n of autogamy f o l l o w i n g the onset of mating r e a c t i v i t y was examined i n c e l l s of i n c r e a s i n g c l o n a l age (1 to 33 f i s s i o n s o l d ) . I n i t i a l l y , autogamy d i d not occur u n t i l about 240 hours a f t e r onset of mating r e a c t i v i t y . a t about 3200 c e l l s / m l . i n n a t u r a l l y s t a r v e d c u l t u r e s . The l a g between onset of mating r e a c t i v i t y and i n i t i a t i o n of autogamy decreased e x p o n e n t i a l l y and reached zero by the 15th c e l l c y c l e . The p e r i o d between 1 and 15 f i s s i o n s can thus be regarded as the immature p e r i o d f o r autogamy s i n c e autogamy does not begin u n t i l mating r e a c t i v i t y has f i r s t been i n i t i a t e d . In o l d e r c e l l s , i n i t i a t i o n of autogamy occurs p r i o r to or c o i n c i d e n t with the development of mating r e a c t i v i t y . T h i s study i n d i c a t e s t h a t there i s no a b s o l u t e immature p e r i o d , but r a t h e r a tendency f o r c e l l s to i n i t i a t e autogamy at p r o g r e s s i v e l y lower l e v e l s of s t a r v a t i o n as c l o n a l age i n c r e a s e s . I t was found t h a t u n t i l about 17 f i s s i o n s , the p o i n t a t which 50% of the c e l l s had completed autogamy was reached no e a r l i e r than the p o i n t a t which f u l l s t a t i o n a r y phase c e l l d e n s i t y was a t t a i n e d (at l e a s t 5000 c e l l s / m l . ) . C o n c u r r e n t l y , -31-the median p o i n t of commitment to autogamy does not f a l l below a c e l l d e n s i t y of 5000 c e l l s / m l . u n t i l about 15 f i s s i o n s . During t h i s i n t e r v a l c o n j u g a t i o n takes p l a c e more r e a d i l y than autogamy s i n c e l e s s s t a r v a t i o n i s r e q u i r e d to induce the former than the l a t t e r . The d u r a t i o n of the immature p e r i o d f o r autogamy has been estimated to be 15 to 20 c e l l c y c l e s by v a r i o u s observers (Sonneborn, 1957, 1974; Miwa and Hiwatashi, 1980; Bleyman, 1971). T h i s study r e v e a l s the b a s i s f o r much of the v a r i a t i o n i n the observed d u r a t i o n of the immature p e r i o d , f o r c e l l s i n i t i a t e autogamy a t p r o g r e s s i v e l y lower l e v e l s of s t a r v a t i o n as c l o n a l age i n c r e a s e s . The observed immature p e r i o d depends on the time ela p s e d between onset of s t a r v a t i o n and s c o r i n g f o r presence of autogamy. Sonneborn (1957, 1974) s t a t e d t h a t autogamy i n P. t e t r a u r e l i a cannot be induced by s t a r v a t i o n d u r i n g the f i r s t 12 t o 15 c e l l d i v i s i o n s a f t e r the beginning of a c l o n a l c y c l e , r e g a r d l e s s of whether the c l o n a l c y c l e i s begun by autogamy or c o n j u g a t i o n . I have shown t h a t autogamy i s i n d u c i b l e even i n very young c e l l s . In young c e l l s autogamy occurs o n l y a f t e r severe s t a r v a t i o n . As c e l l s become o l d e r , autogamy occurs sooner and sooner a f t e r the onset of mating r e a c t i v i t y . The absence of r e p o r t s of autogamy i n young c e l l s i n e a r l i e r s t u d i e s i s l i k e l y the r e s u l t of o b s e r v a t i o n a l p e r i o d s of inadequate d u r a t i o n . The c o n s i s t e n t e x p o n e n t i a l decrease i n l a g between the onset of mating r e a c t i v i t y and i n i t i a t i o n of autogamy with i n c r e a s i n g c l o n a l age suggests t h a t immaturity f o r autogamy i n -32-P. t e t r a u r e l i a i s governed by a d i l u t a b l e f a c t o r . Clones of some s p e c i e s of Paramecium have, a f t e r c o n j u g a t i o n , a p e r i o d of immaturity d u r i n g which the c e l l s cannot mate (Sonneborn, 1957). Miwa e t a l . (1975) found t h a t i n three d i f f e r e n t groups of Paramecium s p e c i e s ? the cytoplasm of immature c e l l s c o n t a i n s a p r o t e i n of MW 10,000 d. T h i s p r o t e i n , c a l l e d immaturin (Haga and Hiwatashi, 1981), r e p r e s s e s mating r e a c t i v i t y when i n j e c t e d i n t o s e x u a l l y mature c e l l s . Although P. t e t r a u r e l i a has no immaturity p e r i o d f o r c o n j u g a t i o n , i t does respond t o immaturity substances from P. p r i m a u r e l i a and P. caudatum (Miwa, 1979). That i s , when the immature cytoplasm of the l a t t e r two s p e c i e s i s m i c r o i n j e c t e d i n t o mature P. t e t r a u r e l i a , mating r e a c t i v i t y i s not developed f o r a p e r i o d of 10 to 15 c e l l c y c l e s . I t seems l i k e l y , t h e r e f o r e , t h a t P. tetraurelia c e l l s share a common immaturin r e c e p t o r with these s p e c i e s . I f , as Sonneborn (1974) noted, the time l a g between the onset of mating r e a c t i v i t y and onset of autogamy decreases as the m a t u r i t y of the c e l l s i n c r e a s e s , i t i s p o s s i b l e t h a t with each c e l l c y c l e immaturin- l i k e substances are p r o g r e s s i v e l y d i l u t e d so t h a t the p e r i o d of immaturity f o r autogamy becomes p r o g r e s s i v e l y s h o r t e r . I f an immaturin- l i k e substance occurs i n P. t e t r a u r e l i a , i t must c o n t r o l autogamy not c o n j u g a t i o n . Since P. tetraurelia c e l l s respond to immaturin from P. caudatum (Miwa, 1979), P. t e t r a u r e l i a might have immaturin- l i k e substances which a c t i n a d i f f e r e n t manner than what i s observed i n P. caudatum, -33-namely to i n h i b i t autogamy. I n t e r p r e t a t i o n of the p a t t e r n of decrease i n l a g between mating r e a c t i v i t y and autogamy i s not a simple matter. Miwa e t a l . (1975) found t h a t the e f f e c t of immaturin o f t e n l a s t e d f o r more than 15 f i s s i o n s when i n j e c t e d i n t o mating r e a c t i v e (mature) c e l l s . They reasoned t h a t i f simple d i l u t i o n with each s u c c e s s i v e c e l l c y c l e was r e s p o n s i b l e f o r the observed s u p p r e s s i o n of mating r e a c t i v i t y , then 15 f i s s i o n s a f t e r the -15 i n j e c t i o n t here should be 2 of the amount i n i t i a l l y i n j e c t e d or an amount f a r lower than the a c t u a l c o n c e n t r a t i o n found i n an immature c e l l or one t h a t i s unable to mate (Miwa e t a l . , 1975). For these g i v e n reasons, they propose that pre- e x i s t i n g immaturity substances s t i m u l a t e f u r t h e r s y n t h e s i s of the same substances i n each c e l l c y c l e . Hence, immaturln l e v e l s g r a d u a l l y decrease with succeeding f i s s i o n s . Miwa e t a l . suggest t h a t the amount of immaturity substances a t v a r i o u s stages of immaturity c o u l d be measured to v e r i f y the hypothesis l a i d out i n t h e i r paper. A n a l y s i s of the k i n e t i c s of the r e s u l t s of t h i s study shows a f i r s t order e x p o n e n t i a l decrease i n immaturln c o n c e n t r a t i o n a t a r a t e of 21% per c e l l c y c l e . T h i s i s c o n s i s t e n t with the idea of a u t o c a t a l y t i c a c t i v a t i o n of immaturin s y n t h e s i s combined with growth d r i v e n d i l u t i o n . T h i s e x p l a n a t i o n would account f o r the p l a t e a u e v e n t u a l l y reached which l i k e l y marks the end p o i n t of immaturin- l i k e p r o t e i n s y n t h e s i s or the c r i t i c a l c o n c e n t r a t i o n of the substance below which autogamy occurs without p r i o r - 3 4 -i I development of mating r e a c t i v i t y . I The problem presented by the hypothesis of a u t o c a t a l y t i c a c t i v a t i o n of immaturin s y n t h e s i s i s t h a t the f i r s t order decay i n immaturin c o n c e n t r a t i o n suggested by t h i s study should r e s u l t i n a l i n e a r decrease i n l a g with i n c r e a s i n g c l o n a l age i f a f i r s t order decay i s assumed to occur d u r i n g the s t a r v a t i o n p e r i o d . On the other hand, a l i n e a r decrease i n immaturin c o n c e n t r a t i o n r e s u l t s i n a f i r s t order decrease i n the l a g p e r i o d . I t i s d i f f i c u l t to see how a l i n e a r decay p a t t e r n of immaturin with i n c r e a s i n g c l o n a l age c o u l d be o b t a i n e d . Kosciusko and Koizumi (1983) have used a d i f f e r e n t approach to study immaturity f o r autogamy. They t r a n s p l a n t e d macronuclear karyoplasm from young c l o n a l age donors to pre- autogamous r e c i p i e n t s . I n j e c t i o n of macronuclear karyoplasm had no e f f e c t on the r a t e and t i m i n g of autogamy. On the other hand, when macronuclear karyoplasm from p r e - autogamous donors was t r a n s p l a n t e d to young r e c i p i e n t s , the d u r a t i o n of the immature p e r i o d was s i g n i f i c a n t l y reduced. F u r t h e r , removal of p a r t o f, the macronucleus l e d to r e d u c t i o n of the immature p e r i o d s u g g e s t i n g t h a t c l o n a l age i s determined by the number of rounds of macronuclear DNA s y n t h e s i s t h a t have taken plac e s i n c e the l a s t f e r t i l i z a t i o n (Mikami and Koizumi, 1983). These o b s e r v a t i o n s are d i f f i c u l t to r e c o n c i l e with my r e s u l t s which imply t h a t a c y t o p l a s m i c f a c t o r Is r e s p o n s i b l e f o r the t i m i n g of occurrence of autogamy. -35-The immature p e r i o d f o r autogamy l i e s wholly w i t h i n the mature p e r i o d f o r c o n j u g a t i o n . The p e r i o d of m a t u r i t y f o r c o n j u g a t i o n begins a f t e r f e r t i l i z a t i o n and p e r s i s t s f o r 25 c e l l c y c l e s , about 10% of the t o t a l l i f e span. C e l l s o l d e r than 25 f i s s i o n s do not show any mating r e a c t i v i t y when allowed to s t a r v e n a t u r a l l y . Throughout t h i s i n t e r v a l mating r e a c t i v i t y develops at a constant l e v e l of s t a r v a t i o n when c u l t u r e s reach a d e n s i t y of approximately 3500 c e l l s / m l . During the i n t e r v a l between 15 and 25 f i s s i o n s autogamy and c o n j u g a t i o n can occur s i m u l t a n e o u s l y i n d i f f e r e n t c e l l s of the p o p u l a t i o n , marking the end of the immature p e r i o d f o r autogamy. T h i s t r a n s i t i o n a l p e r i o d i s terminated by t o t a l l o s s of mating r e a c t i v i t y a t a c l o n a l age of about 25 c e l l c y c l e s . Senescence begins with the end of m a t u r i t y f o r c o n j u g a t i o n a t about 25 f i s s i o n s . From t h i s p o i n t on, a l l c e l l s become committed to autogamy at a c e l l d e n s i t y t h a t i s l o v e r than t h a t r e q u i r e d f o r the onset of mating r e a c t i v i t y . The c e l l c y c l e experiments d i s c u s s e d above e s t a b l i s h e d t h a t commitment to autogamy i s a s s o c i a t e d with l o s s of mating r e a c t i v i t y . Hence, i f c e l l s become committed to autogamy a t a c e l l d e n s i t y t h a t i s lower than t h a t r e q u i r e d f o r the onset of mating r e a c t i v i t y , c o n j u g a t i o n cannot take p l a c e . Sonneborn (1974) s t a t e s t h a t mating does not occur u n t i l a f t e r autogamy i s completed i n these c e l l s . My experiments show t h a t c e l l s do not become mating r e a c t i v e a f t e r completion of autogamy unless they are r e f e d . Autogamous c u l t u r e s were r e f e d by adding f r e s h medium i n v a r i o u s -36-amounts (0.5% to 5% of i n i t i a l volume). Mating r e a c t i v i t y developed i n a l l the r e f e d c u l t u r e s as the added food was exhausted. No mating r e a c t i v i t y appeared i n the unfed c o n t r o l c u l t u r e . The p e r i o d of senescence comprises almost 90% of the c l o n a l l i f e span. Autogamy occurs r e a d i l y u n t i l about 100 f i s s i o n s a f t e r which the process of s e l f - f e r t i l i z a t i o n becomes harder to induce as the c e l l s approach the end of t h e i r c l o n a l l i f e (Sonneborn, 1974). Sonneborn's use of the term "senescence" to d e s c r i b e the p o r t i o n of the l i f e h i s t o r y of P. t e t r a u r e l i a i n which mating does not normally occur may be somewhat m i s l e a d i n g . My abrupt downshift experiments and those of Beisson and C a p d e v i l l e (1966) have shown t h a t when l o g phase c u l t u r e s 30 to 35 f i s s i o n s o l d are washed i n D r y l ' s b u f f e r the m a j o r i t y of c e l l s (approximately 70%) develop mating r e a c t i v i t y a f t e r 3.0 to 3.5 hours. These c e l l s are beyond the 25 f i s s i o n p e r i o d f o r c o n j u g a t i o n , but are a b l e t o mate when s u b j e c t e d to c o n d i t i o n s t h a t a c t i v a t e both s e x u a l pathways s i m u l t a n e o u s l y . In both s e t s of experiments, there was a subsequent sharp drop i n mating r e a c t i v i t y which was followed by a sharp r i s e i n the percentage of c e l l s completing autogamy, beginning approximately s i x hours a f t e r washing. E s t i m a t i o n of the t i m i n g of commitment to autogamy shows t h a t l o s s of mating r e a c t i v i t y c o i n c i d e s with i n i t i a t i o n of autogamy. The term "senescence" thus seems i n a p p r o p r i a t e because s u r v i v a l and f e r t i l i t y f o l l o w i n g both c o n j u g a t i o n and autogamy i s -37-100% u n t i l long a f t e r the s t a r t of the "senescent' 1 p e r i o d . T h e r e f o r e , the term might be more s u i t a b l y a s s i g n e d to t h a t p e r i o d of the organism's c l o n a l l i f e d u r i n g which autogamy i s d i f f i c u l t to induce and the frequency of l e t h a l i t y i n c r e a s e s due to accumulation of mutations (Sonneborn, 1974). The term "mature p e r i o d f o r autogamy" co u l d r e p l a c e Sonneborn*s "senescence" f o r t h a t p o r t i o n of Paramecium's c l o n a l l i f e h i s t o r y where autogamy occurs r e a d i l y . T h i s study shows t h a t autogamy Is the " p r e f e r r e d " s e x u a l process except i n young c e l l s . F i r s t , depending on t h e i r age, c e l l s do not need to be as s t a r v e d to enter the autogamy pathway as they do to become mating r e a c t i v e . Second, commitment to autogamy shuts o f f mating r e a c t i v i t y . T h i r d , a l l c e l l s , r e g a r d l e s s of c l o n a l age, are competent to undergo autogamy while o n l y young c e l l s are competent to p a r t i c i p a t e i n c o n j u g a t i o n . As c l o n a l age i n c r e a s e s , the time between the onset of mating r e a c t i v i t y and autogamy d e c r e a s e s / and, iw,.older c e l l s , autogamy e v e n t u a l l y precedes and prevents c o n j u g a t i o n . The r e s u l t s of t h i s study need to be placed i n the broader context of the breeding s t r a t e g i e s of the P. auzelia s p e c i e s complex. Some P. a u r e l i a s p e c i e s are extremely inbred (Landis, 1986). These p o p u l a t i o n s are c h a r a c t e r i z e d as having s h o r t p e r i o d s of immaturity, i f any, and s h o r t p e r i o d s of m a t u r i t y f o r c o n j u g a t i o n ( L a n d i s , 1986, 1988; Sonneborn, 1957). The s h o r t p e r i o d of immaturity presumably decreases the p r o b a b i l i t y of d i s t a n t l y r e l a t e d i n d i v i d u a l s ( c e l l s from very d i f f e r e n t c l o n a l -38-p o p u l a t i o n s ) mating, s i n c e time f o r d i s p e r s a l i s not allowed ( L a n d i s , 1986). Short p e r i o d s of m a t u r i t y f o r c o n j u g a t i o n encourage c o n j u g a t i o n w i t h i n a p o p u l a t i o n s i n c e the time a l l o t t e d to t h i s a c t i v i t y i s b r i e f ( L a n d i s , 1986). The remainder of the l i f e h i s t o r y i s devoted to autogamy, the primary consequence of which i s homozygosity at a l l l o c i . -S e l e c t i o n w i l l clump together i n t e r a c t i n g a l l e l e s a t d i f f e r e n t l o c i t h a t have become mutu a l l y adapted. Inbreeding, i n t u r n , w i l l m aintain such a l l e l i c complexes w i t h i n a c l o n e . T h e r e f o r e , over a s u f f i c i e n t l e n g t h of time, c l o n a l p o p u l a t i o n s become g e n e t i c a l l y i s o l a t e d from each other. For example, c o n j u g a t i o n between s t o c k s of d i v e r s e o r i g i n r e s u l t s i n high l e t h a l i t y i n P. t e t r a u r e l i a , while c o n j u g a t i o n w i t h i n s t o c k s produces v i r t u a l l y 100% f e r t i l i t y . The advantage of t h i s phenomenon i s t h a t each p o p u l a t i o n becomes a unique s e t of h i g h l y adapted i n d i v i d u a l s . C l o s e l y r e l a t e d s p e c i e s can thus c o h a b i t a s i n g l e r e g i o n without competing f o r the same r e s o u r c e . The disadvantage i s t h a t g e n e t i c d i v e r s i t y w i t h i n s t o c k s of P. t e t r a u r e l i a i s v e r y low due to occurrence of autogamy and lack of c o n j u g a t i o n between p o p u l a t i o n s . On the other hand, the r a t e of s p e c i a t i o n i s high s i n c e p o p u l a t i o n s become both g e n e t i c a l l y and r e p r o d u c t i v e l y i s o l a t e d from each other thereby becoming i n c i p i e n t b i o l o g i c a l s p e c i e s . The a u r e l i a complex was l i k e l y once a s i n g l e s p e c i e s (P. a u r e l i a ) which now comprises a t l e a s t 15 separate s p e c i e s . The complex r e p r e s e n t s a range of breeding s t r a t e g i e s with some s p e c i e s being l e s s s t r o n g l y i n b r e e d i n g than P. t e t r a u r e l i a . The -39-prevalence of autogamy and the r e s t r i c t i o n of c o n j u g a t i o n t o c e l l s w i t h i n a p o p u l a t i o n were probably s i g n i f i c a n t f a c t o r s i n the development of t h i s s i b l i n g s p e c i e s complex. The P. b u r s a r i a complex, on the other hand, i s h i g h l y outbreeding. A l l s p e c i e s are capable of Inbreeding - i t i s the degree to which t h i s occurs t h a t makes an organism an outbreeder or an in b r e e d e r . Inbreeding and outbreeding are r e l a t i v e terms. Conjugation between p o p u l a t i o n s i n P. b u r s a r i a s p e c i e s i s encouraged by the long p e r i o d of m a t u r i t y f o r c o n j u g a t i o n , by the long immature p e r i o d i n t h i s s p e c i e s complex, and by the presence of m u l t i p l e mating types. The long p e r i o d of immaturity f o r c o n j u g a t i o n presumably a l l o w s f o r d i s p e r s a l of i n d i v i d u a l s to areas occupied by d i f f e r e n t p o p u l a t i o n s before mating. The long p e r i o d of m a t u r i t y f o r c o n j u g a t i o n g i v e s ample time f o r these d i s t a n t l y r e l a t e d i n d i v i d u a l s t o mate when they have met. The m u l t i p l e mating types of P. b u r s a r i a i n c r e a s e the chances of mating between c e l l s of d i f f e r e n t p o p u l a t i o n s . Members of the P. b u r s a r i a complex, i n c o n t r a s t to the P. aurelia complex, a l s o do not undergo autogamy. Consequently, the r a t e of g e n e t i c divergence between p o p u l a t i o n s i s lower and f e r t i l i t y i n i n t e r s t o c k c r o s s e s i s hi g h . Conversely, autogamy leads t o immediate e x p r e s s i o n of any g e n e t i c v a r i a b i l i t y w i t h i n the p o p u l a t i o n . Newly generated mutant a l l e l e s would be expressed, and immediately s u b j e c t e d to s e l e c t i v e p r e s s u r e , r e s u l t i n g i n maximum i n t e r - p o p u l a t i o n g e n e t i c d i f f e r e n t i a t i o n with minimum i n t r a - p o p u l a t i o n g e n e t i c v a r i a t i o n . In the P. a u r e l i a complex, the occurrence of o n l y two mating types r e s t r i c t s mating r e l a t i v e t o the m u l t i p l e mating type system of the P. b u r s a r i a complex. P. t e t r a u r e l i a , the extreme inbreeder of the P. a u r e l i a complex, has c a r r i e d t h i s t r e n d even f u r t h e r . The g r e a t m a j o r i t y of the n a t u r a l i s o l a t e s of t h i s s p e c i e s have been of a s i n g l e mating type (odd) ( L a n d i s , 1988). T h i s makes c o n j u g a t i o n v i r t u a l l y impossible under n a t u r a l c o n d i t i o n s . The presence of a p e r i o d of m a t u r i t y f o r c o n j u g a t i o n and a p e r i o d of immaturity f o r autogamy suggest that a t some p o i n t c o n j u g a t i o n must have been u s e f u l , and the two mating types probably e x i s t e d i n more equal frequency. The s c a r c i t y of the even mating type i n the w i l d may be due to the observed d i f f e r e n c e (12%, p<0.001) i n growth r a t e s between the two mating types ( F i g . 18). Even i f the two mating types of P. tetraurelia e x i s t e d i n equal frequency today, autogamy would s t i l l be the primary s e x u a l process s i n c e the mature p e r i o d f o r c o n j u g a t i o n comprises o n l y ten percent of the c l o n a l l i f e span. T h i s i m p l i e s t h a t P. t e t r a u r e l i a has long been a l a r g e l y a u t o m i c t i c organism (one that r e l i e s h e a v i l y on s e l f i n g to produce progeny). The frequency of c o n j u g a t i o n i n nature i s now near zero s u g g e s t i n g t h a t P. t e t r a u r e l i a has evolved i n t o a wholly a u t o m i c t i c organism. As a r e s u l t , P. t e t r a u r e l i a behaves l i k e a non- m i c t i c h a p l o i d s p e c i e s s i n c e autogamy e n f o r c e s complete homozygosity and o u t c r o s s i n g between s t o c k s i s n e a r l y i m p o s s i b l e . •• -41-We must then ask why autogamy retains such a prominent place in the l i f e h istory of Paramecium t e t r a u r e l i a . The answer is ph y s i o l o g i c a l . The organism's short l i f e span requires frequent nuclear reorganization (meiosis, f e r t i l i z a t i o n and macronuclear development) to produce a young macronucleus and the attendant rejuvenation. Aging in c i l i a t e s i s e s s e n t i a l l y a macronuclear phenomenon (Aufderheide, 1987). It i s associated with decreases in growth rate and general vigor and i s accompanied by decrease in DNA repair a c t i v i t y and accumulation of l e t h a l mutations (Fukushima, 1975; Smith- Sonneborn, 1981; Sonneborn and Schneller, 1960). Paramecium i s thus dependent on autogamy to produce new raacronuclei at frequent intervals even when there is l i t t l e or no genetic d i v e r s i t y within populations. The entire breeding strategy is strongly conservative and suggests that the organism i s highly adapted to a c l o s e l y defined niche in a very stable environment. -42-C L O N A L LIFE HISTORY Conjugat ion 1 " \ k 1 1 015 25 5 0 100 2 0 0 fissions CLONAL AGE FIGURE 1: Clonal l i f e h i s t o r y of Paramecium t e t r a u r e l i a shoving maturity for conjugat ion, immaturity for autogamy, and senescence. - 4 3 -4 0 >-> I— LU Q: O 20 o o o 5 0 o J L J I I I I L 0 10 20 30 4 0 TIME (min) 50 FIGURE 2: S t a b i l i z a t i o n of mating r e a c t i v i t y i n b u f f e r . F i l l e d c i r c l e s : f i r s t s e t of o b s e r v a t i o n s ; open c i r c l e s : second s e t of o b s e r v a t i o n s . -44-4 0 0 0 2 3 0 0 0 LU Q_ co 2 0 0 0 1 0 0 0 -o o o o o -LU u O 8 0 UJ CD 6 0 i< z LU 4 0 £ LU CL 20 06-O-0—-o-1—1—1—1—1—1—1—1— 0 2 4 6 8 10 12 TIME (hours) 0 FIGURE 3: Increase i n c e l l number and onset of mating r e a c t i v i t y i n a n a t u r a l l y s t a r v e d c u l t u r e of unknown age. F i l l e d c i r c l e s : c e l l d e n s i t y ; open c i r c l e s : percentage of c e l l s showing mating r e a c t i v i t y . -45-1 2 3 4 5 Time after downshift (h) FIGURE 4: Mating r e a c t i v i t y onset in D r y l ' s buf fer . Open c i r c l e s set of observations; f i l l e d c i r c l e s : second set of observat ions . -46-5 0 100 Stage in cell cycle(%) FIGURE 5: Mating r e a c t i v i t y onset as a f u n c t i o n of c e l l c y c l e stage occupied by c e l l s upon n u t r i e n t downshift. -47-> u 03 L_ D) C ru 6 0 5 0 4 0 3 0 C9 „ g> 20 c o 1 0 -o o o# • • ° o o o Oo o o o ^ i i i i i i i i i i i i i T i i 0 5 10 15 20 Time a f te r downshift (h) FIGURE 6: P e r s i s t e n c e of mating r e a c t i v i t y i n immature c e l l s . Open c i r c l e s : n a t u r a l l y s t a r v e d c e l l s ; f i l l e d c i r c l e s : c e l l s s u b j e c t e d to abrupt downshift and maintained i n b u f f e r . -48-4 0 0 0 o 2 0 0 0 U Auto o—©100 Mating reactivity j l mi ^ f r i i m i Q) 03 5 0 c U 0 4 6 8 Time (h) 10 12 F I G U R E 7: Occurrence of autogamy i n n a t u r a l l y s t a r v e d "senescent" c e l l s , mature for autogamy. Open c i r c l e s : c e l l d e n s i t y ; c i r c l e s wi th c e n t r a l d o t : percentage of c e l l s showing macronuc lear f r a g m e n t a t i o n ; f i l l e d c i r c l e s : percentage of c e l l s showing mat ing r e a c t i v i t y ; c r o s s e s : e s t i m a t e d percentage of c e l l s c o m p l e t i n g the p r e - autogamous f i s s i o n . - 4 9 -1 0 0 Ci) D) CU Mat ing R e a c t i v i t y A u t o g a m y O J L 0 o , y o o o o / J I I L J L 5 10 15 T i m e a f t e r d o w n s h i f t (h) FIGURE 8: Occurrence of autogamy and mating r e a c t i v i t y i n an a b r u p t l y s t a r v e d mature c u l t u r e . F i l l e d c i r c l e s : percentage of c o n t r o l mating r e a c t i v i t y ; open c i r c l e s : percentage of c e l l s showing macronuclear fragmentation; c r o s s e s : estimated percentage of c e l l s completing the pre- autogamous f i s s i o n . -50-4000 3000-LU CO LU 2000-<~> 1000T-TIME (hours) FIGURE 9 : Mating r e a c t i v i t y and occurrence of autogamy in a cul ture containing both mature and immature c e l l s . F i l l e d c i r c l e s : mating r e a c t i v i t y ; open c i r c l e s with centra l dot: percentage of c e l l s showing macronuclear fragmentation; open c i r c l e s : c e l l dens i ty ; crosses: estimated f r a c t i o n of c e l l s completing the pre- autogamous f i s s i o n . -51-> u CU C9 CD c cu E C9 O) CU c U L_ P— — O \ \ __Q Q Immature ° o Mature \ * Autogamy o 6 0 50 4 0 30 20 10 0 -4 -3 -2 - 1 0 1 2 3 4 5 Hours after pre-autogamous fission FIGURE 10: Mating r e a c t i v i t y during the f i r s t and second c e l l cycles after nutr ient downshift. Open c i r c l e s : immature c e l l s ; f i l l e d c i r c l e s : mature c e l l s . -52-TIME (hr) FIGURE 11: Percent autogamy as a f u n c t i o n of time a f t e r mating r e a c t i v i t y onset i n mature c u l t u r e s of i n c r e a s i n g c l o n a l age. Open c i r c l e s : 3 f i s s i o n s o l d ; f i l l e d c i r c l e s : 5 f i s s i o n s o l d ; open diamonds: 7 f i s s i o n s o l d ; open squares: 9 f i s s i o n s o l d ; c r o s s e s : 11 f i s s i o n s o l d ; f i l l e d squares: 13 f i s s i o n s o l d . -53-TIME (hr) FIGURE 12: Percent autogamy as a function of time af ter mating r e a c t i v i t y onset in mature cul tures of increas ing c lona l age. F i l l e d c i r c l e s : 15 f i s s i ons o l d ; open c i r c l e s : 17 f i s s i ons o l d ; crosses: 19 f i s s ions o l d ; open diamonds: 21 f i s s ions o ld ; f i l l e d c i r c l e s : 23 f i s s ions o l d ; open squares: 25 f i s s ions o l d . - 5 4 -o CD < 200 -150 100 0 5 10 15 20 CLONAL AGE (fissions) 25 FIGURE 13: Time between i n i t i a t i o n of mating r e a c t i v i t y and i n i t i a t i o n of macronuclear fragmentation i n s t a t i o n a r y phase c u l t u r e s as a f u n c t i o n of c l o n a l age. -55-3 4 CELLS/ML x 1000 FIGURE 14: Percent autogamy as a f u n c t i o n of c e l l d e n s i t y i n mature c u l t u r e s of i n c r e a s i n g c l o n a l age. Open c i r c l e s : 9 f i s s i o n s o l d ; f i l l e d c i r c l e s : 11 f i s s i o n s o l d ; c r o s s e s : 13 f i s s i o n s o l d ; f i l l e d s quares: 15 f i s s i o n s o l d ; open squares: 17 f i s s i o n s o l d ; open diamonds: 19 f i s s i o n s o l d . -56-FIGURE 15: Percent autogamy as a f u n c t i o n of c e l l d e n s i t y i n mature c u l t u r e s of i n c r e a s i n g c l o n a l , a g e . Open c i r c l e s : 21 f i s s i o n s o l d ; c r o s s e s : 23 f i s s i o n s o l d ; f i l l e d c i r c l e s : 25 f i s s i o n s o l d ; open squares: 27 f i s s i o n s o l d ; f i l l e d squares: 29 f i s s i o n s o l d ; open diamonds: 31 f i s s i o n s o l d . -57-1 2 3 4 5 CELLS/ML x 1000 FIGURE 16: Percent autogamy as a function of c e l l densi ty in mature cul tures of increasing c l o n a l age. F i l l e d c i r c l e s : 33 f i s s ions o l d ; open c i r c l e s : 35 f i s s i ons o l d ; f i l l e d squares: 37 f i s s ions o l d ; crosses: 39 f i s s ions o l d ; open diamonds: 41 f i s s ions o l d ; f i l l e d diamonds: 41 f i s s i ons old (27 C c o n t r o l ) . -58-6 0 0 0 5000 LJ 4 0 0 0 ^ 3 0 0 0 ©©€)© + + UJ 2000 U 1000 0 o * ° ° o + O + _ ooo o o + + J I I I L J L O 10 20 30 40 CLONAL AGE (fissions) FIGURE 17: C e l l d e n s i t y at autogamy and i n i t i a t i o n of mating r e a c t i v i t y as a f u n c t i o n of c l o n a l age. Open c i r c l e s : c e l l d e n s i t y when 50% of c e l l s showed macronuclear fragmentation; f i l l e d c i r c l e s : c e l l d e n s i t y a t onset of mating r e a c t i v i t y ; c r o s s e s : estimated c e l l d e n s i t y a t median time of commitment to autogamy. -59-CO LO CO U. LU > < _ J ZD ZD - • 100 • • • • • • • n 6 0 H B 20 • T I I i i i i i i i i 2 4 6 8 10 DAY OF CULTURE FIGURE 18: Comparison of growth r a t e s of even and odd mating types. F i l l e d squares: odd mating type; open squares: even mating type. -60-BIBLIOGRAPHY Aufderheide, K.J. 1987. C l o n a l aging i n Paramecium t e t r a u r e l i a . I I . Evidence of f u n c t i o n a l changes i n the macronucleus with age. Mech Ageing Develop. 37, 265-279. B e i s s o n , J . and Y. C a p d e v i l l e . 1966. Sur l a nature p o s s i b l e des etapes de d i f f e r e n c i a t i o n conduisant a l'autogomie chez Paramecium a u r e l i a . C R . Acad. S c i . 263, 1258-1261. Berger, J.D. 1986. Autogamy i n Paramecium t e t r a u r e l i a : c e l l c y c l e stage s p e c i f i c d e t e r m i n a t i o n of the p r o c e s s . Exp. C e l l Res. 166, 475-486. Berger, J.D. and AS-L Ching. 1988. The t i m i n g of i n i t i a t i o n of DNA s y n t h e s i s i n Paramecium t e t r a u r e l i a i s e s t a b l i s h e d d u r i n g the preceding c e l l c y c l e as c e l l s become committed t o c e l l d i v i s i o n . Exp. C e l l Res. 174, 355-366. Bleyman, L.K. 1971. Temporal p a t t e r n s i n the c i l i a t e d p r otozoa. In Developmental aspects of the c e l l c y c l e . T.L. Cameron, G.M. P a d i l i a , and A.S. Zimmerman, eds. Academic P r e s s . New York. 67-91. Brock, T.D. 1965. The p u r i f i c a t i o n and c h a r a c t e r i z a t i o n of an i n t e r c e l l u l a r sex- s p e c i f i c mannan p r o t e i n from y e a s t . Proc. N a t l . Acad. S c i . U.S.A. 54, 1104-1112. Ching, AS-L and J.D. Berger. 1986. C o n t r o l of c e l l d i v i s i o n i n Paramecium tetraurelia: e f f e c t s of abrupt changes i n n u t r i e n t l e v e l on accumulation of macronuclear DNA and i n i t i a t i o n of c e l l d i v i s i o n . Exp. C e l l Res. 167, 191-202. C r a n d a l l , M., L.M. Lawrence and R.H. Saunders. 1974. Molecular complementarity of yeast g l y c o p r o t e i n mating f a c t o r s . Proc. N a t l . Acad. S c i . U.S.A. 71, 26-29. Fukushima, S. 1975. C l o n a l age and the p r o p o r t i o n of d e f e c t i v e progeny a f t e r autogamy i n Paramecium a u r e l i a . G e n e t i c s . 79, 377-382. Haga, N. and K. Hiwatashi. 1981. A p r o t e i n c a l l e d immaturin c o n t r o l l i n g s e x u a l immaturity i n Paramecium caudatum. Nature (London). 289, 177-179. Hiwatashi, K. 1969b. Paramecium. In F e r t i l i z a t i o n . C.B. Metz and A. Monroy. eds. Academic P r e s s . New York. V o l . 2, 255-293. -61-Kitamura, A. and K. Hiwatashi. 1978. Are sugar r e s i d u e s i n v o l v e d i n the s p e c i f i c c e l l r e c o g n i t i o n of mating i n Paramecium? J . Exp. Z o o l . 203, 99-108. K l a s s , M.R. and J . Smith-Sonneborn. 1976. St u d i e s on DNA content, RNA s y n t h e s i s and DNA template a c t i v i t y i n ageing c e l l s of Paramecium aurelia. Exp. C e l l Res. 98, 63-72. Kosciuszko, H. and S. Koizumi. 1983. In d u c t i o n of autogamy by t r a n s f e r of macronuclear chromatin i n Paramecium t e t r a u r e l i a . Exp. C e l l Res. 146, 436-438. L a n d i s , W.G. 1986. The i n t e r p l a y among ecology, breeding system, and g e n e t i c s i n the Paramecium aurelia and Paramecium bursaria complexes. Progr. P r o t i s t o l . 1, 225-245. La n d i s , W.G. 1988. Ecology. In Paramecium. H.D. G o r t z . ed. S p r i n g e r - V e r l a g . New York. 419-436. Metz, C.B. 1954. Mating substances and the p h y s i o l o g y 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. D.H. Wenrich. ed. Am. Assoc. Adv. S c i . Washington, D.C. 284-334. Mikami, K. and S. Koizumi. 1983. M i c r o s u r g i c a l a n a l y s i s of the c l o n a l age and the c e l l - c y c l e stage r e q u i r e d f o r the onset of autogamy i n Paramecium t e t r a u r e l i a . Dev. B i o l . 100, 127-132. Miwa, I. 1979. Immaturity substances i n Paramecium p r i m a u r e l i a and t h e i r s p e c i f i c i t y . J . C e l l S c i . 38, 193-199. Miwa, I . , N. Haga, and K. Hiw a t a s h i . 1975. Immaturity substances: m a t e r i a l b a s i s f o r immaturity i n Paramecium. J . C e l l S c i . 19, 369-378. Miwa, I. and K. Hiwatashi. 1970. E f f e c t of mitomycin C on the e x p r e s s i o n of mating a b i l i t y i n Paramecium caudatum. Jap. J . Genet. 45, 269-275. Miwa, I . and N. Umehara. 1983. Conjugation between G and G 2 phase c e l l s i n Paramecium t e t r a u r e l i a . J . P r o t o z o a l . 30, 271-274. Miyake, A. and J . Beyer. 1973. C e l l i n t e r a c t i o n by means of s o l u b l e f a c t o r s (gamones) i n c o n j u g a t i o n of Blepharisma intermedium. Exp. C e l l Res. 76, 15-24. Pet e r s o n , E.L. and J.D. Berger. 1976. M u t a t i o n a l blockage of DNA s y n t h e s i s i n Paramecium t e t r a u r e l i a . Can. J . Z o o l . 54, 2089-2097. -62-Rasmussen, L. 1967. E f f e c t s of metabolic i n h i b i t o r s on Paramecium a u r e l i a d u r i n g the c e l l g e n e r a t i o n c y c l e . Exp. C e l l Res. 48, 132-139. Rasmussen, C D . and J.D. Berger. 1984. A gene f u n c t i o n r e q u i r e d f o r c e l l c y c l e p r o g r e s s i o n d u r i n g the G- p o r t i o n of the c e l l c y c l e and f o r maintenance of macronuclear DNA s y n t h e s i s i n Paramecium tetraurelia. Exp. C e l l Res. 155, 593-597. Schwartz, V. and H. M e i s t e r . 1973. Eine A l t e r s v e r a n d e r u n g des Makronucleus von Paramecium. Z. N a t u r f o r s c h . 28c, 232. Schwartz, V. and H. M e i s t e r . 1975. Aging i n Paramecim: S e v e r a l q u a n t i t a t i v e a s p e c t s . Arch. P r o t i s t e n k B i o l . 117, 85-109. Smith-Sonneborn, J . 1981. G e n e t i c s and aging i n protozoa. I n t . Rev. C y t o l . 73, 319-354. Sonneborn, T.M. 1950. Methods i n gen e r a l b i o l o g y and g e n e t i c s of Paramecium a u r e l i a . J . Exp. Z o o l . 113, 87-147. Sonneborn, T.M. 1957. Breeding systems, r e p r o d u c t i v e methods, and s p e c i e s problems i n protozoa. In The s p e c i e s problem. E. Mayr. ed. Am. Assoc. Adv. S c i . Washington, D.C. 155-324. Sonneborn, T.M. 1970. Methods i n Paramecium r e s e a r c h . Methods C e l l P h y s i o l . 4, 241-336. Sonneborn, T.M. 1974. Paramecium aurelia. In Handbook of g e n e t i c s I I . R.C. King. ed. Plenum. New York. 469-594. Sonneborn, T.M. and M.V. S c h n e l l e r . 1960. P h y s i o l o g i c a l b a s i s of a g ing i n Paramecium. In The B i o l o g y of Aging (American I n s t i t u t e of B i o l o g i c a l Science Symposium 6 ) . B.L. S t r e h l e r . ed. Waverly P r e s s . B a l t i m o r e , MD. 283-284. Takagi, Y. and N. Kanazawa. 1982. Age- a s s o c i a t e d change i n macronuclear DNA content i n Paramecium caudaturn. J . C e l l S c i . 54, 137-147. Watanabe, T. 1977. C i l i a r y membranes and mating substances i n Paramecium caudaturn. J . P r o t o z o o l . 24, 426-429. Wiese, L. 1974. Nature of sex s p e c i f i c g l y c o p r o t e i n a g g l u t i n i n s i n Chlamydomonas. Ann. N.Y. Acad. S c i . 234, 383-395. -63-

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0098432/manifest

Comment

Related Items