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Protistology 2 (3), 142-151 (2002)
Protistology
Serotypes in the ciliate Dileptus anser: epigenetic phenomena
Alexander L. Yudin and Zoya I. Uspenskaya
Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russia
Summary
This review presents some data obtained by the authors in their study of the serotype system in the lower ciliate Dileptus anser, a species that has not been explored previously in this aspect. These data show many features similar to those described in serotype systems of higher ciliates, Paramecium and Tetrahymena. At the same time, some of the results do not agree with conventional patterns generally accepted for these classical objects. The authors discuss the data obtained in dilepti in terms of epigenetic variation and inheritance.
Key words: ciliates, Dileptus anser, serotypes, immobilization antigens, non-Mendelian inheritance, serotype transformation, regulation of serotype expression, epigenetic variation and inheritance
In the ciliates Paramecium and Tetrahymena, their serotype systems have long been studied and were revealed to have a diversity of peculiar properties.
The ciliate serotype is principally defined through the use of immunological techniques and depends on a certain class of proteins distributed all over the surface of the external cell membrane and cilia (Beale and Mott, 1962; Doerder, 1981). These proteins are referred to as immobilization antigens, or i-antigens, as ciliates treated with the homologous immune serum (IS) at specific concentrations turned out to be immobilized. So far, functions of i-antigens are not known. Their peculiar feature is their great diversity and variability that were first noted as early as in the 1940s (see one of the first reviews by Beale, 1954). The ciliates that belong to the same clone can express various i-antigens. To illustrate this, in the best studied species of the P. aurelia complex, the genome of each cell contains up to 12 nonlinked genes that code different i-antigens. This gene system operates on the principle of «mutual exclusion»:
at any one time, only one i-antigen is detectable on the cell surface of all genotypically available antigens. In a number of cases, this rule is followed not only by different genes, but also by different alleles of the same gene. Exceptional cases (in which two or more i-antigens are revealed simultaneously on the cell surface) occur very seldom. When cultural conditions are changed or some treatments are used, a replacement of one surface protein with another (of all proteins coded in the cell genome) takes place, which is referred to as the cell serotype transformation. This alteration usually involves most or even all cells in the clone and appears to be totally reversible. Thus, different serotypes are expressed under different conditions of temperature, salinity, feeding, etc., and each set of conditions is characteristic of the expression of a specific i-antigen over some range, these ranges being sometimes more or less overlapping.
Once established, a particular serotype tends to be inherited (the so-called «functional inertia» - Nanney,
© 2002 by Russia, Protistology
1980). More specifically, it is not infrequent that ciliates with the same genotype (for instance, from the same clone) that were cultured under different conditions and, therefore, expressed different serotypes, maintain these specific serotypes through cell generations, when transferred to a «zone of overlapping». In other words, under the same conditions, ciliates of the same genotype are capable of expressing and inheriting more than one distinct phenotype. Therefore, phenomena of epigenetic variation and inheritance are very typical of serotype systems in ciliates. It is to be recalled that one of the first hypothetical models for epigenetic control of characters in Paramecium was proposed to explain peculiarities of the genetic control and inheritance of its serotypes (Delbruck, 1949); this hypothesis provided a basis for numerous subsequent ideas of the kind (reviews: Olenov, 1965; Golubovsky, 1996; Golubovsky and Tchuraev, 1997; Riggs and Porter, 1996; Russo et al., 1996; and some others).
It is generally agreed that the most exciting issues in serotype systems of ciliate are the mechanisms of regulation of the genes coding i-antigens under normal conditions and during serotype transformation, which provide their expression in accordance with the principle of «mutual exclusion» and the tendency of the once established serotype towards inheritance in subsequent cell generations. These problems were the objective of many studies (the review of their current status and the basic literature — Bleyman, 1996). In the most advanced cases, not only surface proteins that determine various serotypes were thoroughly characterized, but also corresponding genes were isolated and sequenced (Preer, 1986; Schmidt, 1996). Nonetheless, the above-mentioned special features of serotype systems in ciliates remain unexplained, even though numerous hypotheses are proposed and verified experimentally (Capdeville, 1979; Finger et al., 1995, 1995/1996; Leeck and Forney, 1996).
It is important that a large body of data on the ciliate serotypes accumulated so far were obtained almost exclusively on a few higher ciliates — 3 to 4 species of the Paramecium aurelia complex and, to a lesser extent, on several other Paramecium species and Tetrahymena thermophila (Bleyman, 1996). Needless to say, these well-developed model objects enable performing modern molecular genetic studies but, at the same time, it makes it difficult to discriminate between more general and more special phenomena and patterns. Therefore, it is tempting to recruit some novel species for studies — it may also provide a fresh knowledge of the serotype systems. For the last few years, serotypes of a lower ciliate, Dileptus anser, have been examined at the Laboratory of Cytology of Unicellular Organisms, Institute of Cytology, Russian Academy of
Sciences, St. Petersburg, and the prime objective of the present paper is to review and to systematize these experiments, the primary emphasis being focused on epigenetic phenomena.
Methods for cultivation, cloning, and crossing of Dileptus anser (Ciliata, Holotricha, Gymnostomatida) (= D. margaritifer - Wirnsberger et al., 1984) at the Laboratory were developed quite recently. D. anser seems to be a very promising laboratory model due to some peculiar features of this ciliate (Yudin et al., 1988). Unfortunately, data on its nuclear apparatus, conjugation, and conjugation cycle are as yet rather scant, although it is the nuclear apparatus that allows to consider D. anser a lower ciliate. Some earlier data on the nuclear apparatus were reviewed by Dragesco (1963). Small spherical micronuclei (6 to 20) divide by mitosis at the beginning of cell division. Immediately afterwards the macronucleus that consists of numerous fragments (the so-called «fragmented», or «pulverized», macronucleus) starts its division. The sexual process has the form of conjugation. Three mating types (I-III) were discovered. Usually, conjugation occurs only between cells of complementary mating types (Yudin and Afon’kin, 1987; Yudin et al., 1988, 1990; Afon’kin, 1990). There is no autogamy in the D. anser life cycle (it is to be recalled that, in Paramecium aurelia species, autogamy settles homozygosity for all genes!). On the whole, the pioneering studies of nuclear behavior during conjugation (Vinnikova, 1974a, 1974b, 1975, 1976; Karadzhan, 1985; Golinska and Afon’kin, 1993) allow a preliminary conclusion that D. anser are diplonts with gametic reduction of the chromosome number and classical (metazoan) meiosis (the same being true of all ciliates — Raikov, 1972).
A set of D. anser clones was used in our experiments, each clone being derived from cells isolated at different time from several ponds of the Leningrad District. The ciliates were cultivated in Prescott’s salt solution at 25°C and fed with Tetrahymena pyriformis (Nikolaeva, 1968). For special purposes, some clones were incubated at 17, 19, 21 or 23°C. Immune sera (ISs) were raised against some of the clones by immunization of rabbits with the whole cell homogenates. The antiserum was referred to as homologous with respect to the clone used for immunization, and as heterologous, for all other clones. The serotype of cells was determined mainly from their immobilization by native ISs (the so-called standard immobilization test). Additionally, a method of indirect immunofluorescence was also periodically used. Occasionally, concentrated IS (specifically, their gamma-globulin fractions) were obtained for tests with reaction of precipitation in agar.
In one of our earlier experiments, 20 D. anser clones of various origin were tested with two ISs obtained
against two of the clones; the total of 38 heterologous combinations «cells — ISs» were thereby tested (Uspenskaya, 1988). The dilepti similar to Paramecium and Tetrahymena showed an effective capability of inducing specific antibodies in immunized rabbits: both the ISs had high titers and produced a quite evident immobilization reaction with homologous cells, which was repeatedly described for Paramecium and Tetrahymena (see reviews by Beale, 1954, 1957). Therefore, it was i-antigens that were responsible for specificity of both ISs. In the total of 33 combinations, ciliates failed to show any immobilization when treated with heterologous ISs; this implies that the tested clones belong to some serotypes different from those of homologous clones. However, in four combinations with one of the ISs and in one combination with the other, a weak immobilization was noted; this might reflect some similarity between the tested serotype and that of the homologous clone. These results were confirmed by immunofluorescence analysis and precipitation reaction in agar. Thus, even with a limited number of different ISs, it was possible to reveal a variety of serotypes in natural populations of D. anser. Later on, with other clones, we found only two or three additional cases of cross-reactions of dilepti with heterologous ISs.
As a matter of fact, this kind of serological crossreactions are described in paramecia. In these ciliates, it is well known that i-antigens coded by different (nonallelic) genes do not usually display cross-reactions, whereas, not infrequently, antigens controlled by alleles of the same gene are more or less similar immunologically (Beale, 1957; Bishop, 1963; Schmidt, 1988).
In D. anser, cross-reactions of heterologous ISs were sometimes observed with clones that were cultivated at different temperatures. Particularly, subclones of the clones 28 and 29 were cultivated, each at 25 and 17°C (the «warm» and «cold» subclones, respectively). They were tested with ISs raised against two subclones of the 5D-clone, a «warm» subclone and a «cold» one (IS 5D-25 and IS 5D-17, respectively). Each of the ISs immobilized only homologous 5D-cells («warm» or «cold», respectively). However, both temperature subclones of the 29 clone turned out to react with both anti-5D ISs. Similarly, both IS 5D-25 and IS 5D-17 immobilized the «warm» 28-cells, whereas only IS 5D-17 immobilized the «cold» 28-cells (Uspenskaya, 1990). In these cases, the observed crossreactions might be explained in two ways. On the one hand, the serotype expressed by the clone 29 both at 17 and at 25°C might be related to both «warm» and «cold» i-antigens of the 5D clone, but it is expressed within a broader temperature range. Or, otherwise, cells of the clones 28 and 29 synthesize two different i-antigens that
are expressed simultaneously at both temperatures — contrary to the principle of mutual exclusion.
As to inheritance of serotypes during agamic reproduction of D. anser, the serotype of each clone usually remained invariable in successive cell generations, if the clone was cultivated under invariable conditions. Some clones were repeatedly tested at yearly intervals and constantly showed nearly 100% of cells immobilized by homologous ISs but without any reactions with heterologous ones.
To examine inheritance of D. anser serotypes during conjugation, we used two independently obtained clones, B and D, that belonged to two complementary mating types I and III. They actively conjugated and had no cross-reactions when tested by cell immobilization, i.e. they showed distinct, well-defined serotypes maintained persistently under the standard conditions of cultivation. The immune serum was produced against each of the clones. Crosses between the clones were made afterwards, cells of one clone being marked with Indian ink to determine and select heterotypic pairs of conjugating cells. After 20-22 h, exconjugant cells separated and were isolated. Unfortunately, we failed to reveal the marker in separated exconjugants and thus to determine their «cytoplasmic origin».
Exconjugant Fj clones were tested twice, using two ISs raised against «parental» clones. The first testing was performed in 30 days after conjugation, while the second one, in 4 months, when the clones became sexually mature (Uspenskaya and Yudin, 2000; Yudin and Uspenskaya, 2000). Dilepti from all F1 clones, when 1 month old, were immobilized each with both ISs, i.e. they had «hybrid» phenotypes. As far as it could be judged from the immobilization reaction, i-antigens of two «parental» types were frequently presented in unequal proportion on the surface of these cells. This is illustrated by the fact that the proportion of cells immobilized by each IS differed significantly in 33 of 44 clones. In 14 clones, dilepti showed a stronger reaction with the anti-B IS, while in 19 clones, with the anti-D IS, thus showing no regular predominance of either i-antigen. The «hybrid» phenotype was further maintained by all these Fj clones until their maturation. Some of the clones were tested by the indirect immunofluorescene method and all of them turned out to be homogenous with respect to their serotypes.
Thus, neither «maternal» inheritance of the studied character, nor the situation that might be similar to the «allelic exclusion» of the «parental» serotypes was observed in F1. We also failed to detect anything like «vegetative assortment» of serotypes in heterozygous clones of Tetrahymena thermophila. On the contrary, the pattern of the serotype inheritance resembled that
of allelic serotypes in Paramecium tetraurelia heterozygotes that have co-expression of both alleles (Beale, 1954, 1957; Preer, 1968; Sommerville, 1970; Finger, 1974; Nanney, 1980; Bleyman, 1996).
The genetic nature of the analyzed difference between the clones B and D serotypes certainly was initially unknown; in the simplest case, it might be a difference for one locus (the allelic difference) or for two or several genetic loci. The absence of serological cross-reactions between the B and D clones indicates prominent immunological differences between corresponding i-antigens and thereby argues in favor of the non-allelic nature of the corresponding serotypes. There was no doubt that the phenotype of the Fj clones was «hybrid». Therefore, if the B and D serotypes were allelic, there was co-expression of the corresponding alleles; but if they were non-allelic, the violation of the rule of mutual exclusion of different loci coding i-antigens had to be assumed. To resolve the alternative, the F2 progeny was to be obtained and analysis of segregation for serotype to be made.
To obtain F2, the F1 clones were crossed not with each other, but with each «parental» clones (the so-called back-crosses). And again, all (!) F2 clones had the «hybrid» phenotype similar to that of the Fj clones. Some F2 clones from various crosses were tested once again in 6 and 9 months after they had been obtained. All of them retained their «hybrid» phenotype (Uspenskaya and Yudin, 2000). Thus, no segregation for the analyzed character was observed in the second hybrid generation, which indicates the non-Mendelian inheritance of the character.
The lack of segregation was an unexpected result. It can be considered a consequence of deviation from the «principle of gamete purity» in the first hybrid generation, which requires an explanation. Interestingly, it is this result, the deviation from the normal Mendelizing of characters (and the absence of segregation in F2, in particular!), that is predicted by the epigene hypothesis (Tchuraev, 1975, 2000). This hypothesis is one of the numerous variants of the concept of the dynamic hereditary memory (Riggs and Porter, 1996). According to Tchuraev, one of the possible results of crosses between the forms differing in epigene-controlled characters is the «absorption effect» (or unification, to be more exact), when all epigenes switch to the same state in F1. As an example, the expressable state of the A locus, if active, is designated as A1, while its inactive state, as A0; the inactive allele in the A1A0 epiheterozygote can be activated by the active one, the A1A0 epiheterozygote being converted to the AjAj epihomozygote. As a result, the Fj individuals will produce only one type of gametes (gametic nuclei, in our case), A1. This will inevitably
affect F2 that will show no segregation. In the case of ciliates, however, the scheme is more complicated. They have been known to be characterized by nuclear heteromorphism (Ossipov, 1981). The reproducible changes in gene activity, which are described by the epigene hypothesis, are most likely to occur at the level of genes of the phenogenetically active ampliploid nucleus (Raikov, 1996), the macronucleus. Meanwhile, the violation of the principle of «gamete purity», whose manifestation is the «blended» inheritance in F2, is to occur at the level of phenogenetically inactive micronuclei and their meiotic products (male and female pronuclei). Therefore, the events that occur in the macronucleus are to somehow affect micronuclear genes. The phenomena that might be considered a sort of predetermination of gametic nuclei in ciliates (specifically, during the serotype inheritance) were described by several authors (Sommerville, 1970).
All the above stimulated our study of the serotype expression in Dileptus clones and, specifically, of the effect of temperature and some other factors. It is well known that, in paramecia and tetrahymenas, most serotypes are relatively stable, if the ciliates are cultivated under standard conditions; however, if cultural conditions are changed, some novel serotype can be expressed. Among numerous factors capable of inducing the serotype transformation, temperature is the most extensively studied (see reviews: Beale, 1957; Finger, 1974; Sonneborn, 1975a, 1975b; Preer, 1989).
When dilepti cultivated at 25°C were transferred to the temperature of17°C, they ceased to be immobilized by IS raised against 25°C ciliates (Uspenskaya, 1990; Uspenskaya and Yudin, 1992). It was suggested that ciliates cultivated at 17°C expressed another serotype, i.e. their surface antigen was replaced by some novel i-antigen.
To determine the dynamics of the serotype transformation in D. anser, four different clones of dilepti were used, as well as four native ISs against these clones. Serotypes of the clones were tested by the reaction of cell immobilization every day after changing the cultivation temperature. On the first day, when no cell division was yet observed, the serotype of ciliates transferred from 25 to 17°C did not differ from the initial one. The proportion of cells that were immobilized by the homologous IS was similar to that of the cells cultivated permanently at 25°C. During the subsequent 2 to 5 days this proportion gradually decreased, and by the 5th day the ciliates showed no response at all to the homologous IS, which indicates the total loss of the initial serotype. Usually, 6 to 8 cell divisions occurred at this time interval. When these ciliates were transferred back to 25°C, the initial serotype (i.e., that characteristic of 25°C) was restored, however, significantly
faster (in 3 days). It also took 6 to 8 cell divisions (Uspenskaya, 1990).
The change of serotype induced by temperature was most clearly shown using two ISs simultaneously, one of the antisera being raised against the 5D subclone cultivated at 17°C, while the other, against the 5D subclone cultivated at 25°C. Each of the ISs immobilized only the homologous cells, certainly demonstrating the change of serotype when the ciliates were transferred from 25 to 17°C and vice versa. The gain of the new serotype and the loss of the old one proceeded at equal rate, but more rapidly at 25°C than at 17°C (Uspenskaya, 1990; Uspenskaya and Yudin, 1992).
Obviously, the change of serotype was due to the synthesis of the new i-antigen and to slowing down of the synthesis of the old i-antigen. In P. aurelia, such shift from one synthesis to the other usually takes 3 to 4 cell divisions (Sommerville, 1969). Also in P. aurelia, at least one doubling of the initial cell number was observed before the serotype transformation and 5-6fold amplification, after completion of the process (Beale, 1957; Preer, 1959). The serotype transformation in T. pyriformis took 3 to 4 days (Grass, 1972). According to other authors, the back and forth transformation between the serotypes H and T in this species took place at a time interval, when 2 to 3 cell divisions occurred, i.e., 6 to 8 hours (Phillips, 1971). In T. thermophila the complete transformation was observed in 2 h (Williams et al., 1985).
The changes of serotype in dilepti, which were observed after transfer of the ciliates into different temperature, turned out to be totally reversible, when the clones were returned to the initial temperature conditions. An immunofluorescent analysis showed that the observed serotype alterations resulted not from the selection of rare cells with a new serotype, but from the gradual change of all cells in the transformed culture. It means that the same cells expressed one serotype at 17°C, and another, at 25°C.
For many serotypes of paramecia and tetra-hymenas the temperature limits of expression of the corresponding i-antigens were defined. For the 5D clone of D. anser we succeeded in determining the upper limit of expression of the «cold» serotype and the lower limit of manifestation of the other, «warm» serotype. When this clone was long cultivated at temperatures intermediate between 17 and 25°C (that is, at 23, 21, and 19°C), the dilepti showed some intermediate levels of immobilization with both anti-17°C and anti-25°C IS. In other words, within this interval of temperatures (19-23°C), both the «cold» and the «warm» serotypes were expressed simultaneously (although in different proportions) and, thereby, the principle of «mutual exclusion» of surface
antigens turned out to be violated (Uspenskaya, 1990; Uspenskaya and Yudin, 1992).
Apart from temperature, all reviews dealing with the ciliate serotypes refer to the homologous IS as a transforming factor (Sonneborn, 1950, 1975a, 1975b; Preer, 1968, 1986; Sommerville, 1970; Finger, 1974; Schmidt, 1988). The transforming effect of the antisera was considered to be due to binding ofthe antibodies with the corresponding surface antigens; therefore, the homologous IS by itself was believed to be capable of transforming a serotype (Sonneborn, 1947; Sonneborn and LeSuer, 1948). With accumulation of new experimental data, homologous antisera started to be considered a destabilizing factor converting the cell to an easily modifiable state (Sonneborn, 1950; Nanney, 1980), the further fate of its serotype depending on conditions of cultivation of the ciliates treated with the IS.
As seen from our experimental data (Uspenskaya and Yudin, 1998a, 1998b), in the D. anser clones treated with the homologous ISs for 1 hr, a massive and unidirectional change of their serotype was observed. During the first 4 days after the treatment, the proportion of cells with the initial serotype decreased and, simultaneously, more and more ciliates appeared, which did not respond to the testing homologous IS and, hence, had another serotype. On the 4th day, more than 90% of all ciliates were not immobilized with this testing IS. Thereafter, the reverse process was observed: the ciliates with the initial serotype progressively increased in numbers, and by the 7th day the culture regained the serotype that it had prior to the treatment. In no case the stably transformed cells (i.e., those inheriting the new serotype) were obtained; this was confirmed by an immunofluorescent analysis: the dynamics of fluorescence in stained ciliates had the same regularities. In cells treated with the homologous IS, fluorescence was very bright on the first one or on the first two days - it was identical to that in control. Thereafter, it gradually decreased and totally disappeared by the 4th day. Thus, only 3 or 5 cells with a very weak fluorescence were found in a sample of 50 ciliates at the 4th day of observation.
Curiously, in subclones cultivated at 17°C, the homologous IS induced the transient appearance of not any, but specifically the «warm» serotype that was characteristic of the subclones of this clone cultivated at 25°C and never appeared in similar subclones at 17°C. Similarly, not any, but only the «cold» serotype was revealed in the subclones that were cultivated at 25°C (Uspenskaya and Yudin, 1998a, 1998b). In other words, the homologous ISs induced expression of the «warm» and «cold» serotype, whose appearance and steady maintenance were determined, according to our data, by temperature of cultivation. It is this fact that might
account for temporary, transient changes of the serotype, as the subclones treated with the homologous ISs were cultivated at the temperature constant for each subclone.
It can be thought that the induced serotype (the «warm» one in the ciliates at 17°C and the «cold» one in the dilepti at 25°C) could not be completely fixed, as the temperature was unfavorable for its maintenance. Nevertheless, this «temperature» antigen did start to be synthesized and expressed at an inappropriate temperature. This might indicate a peculiar sensitivity of the corresponding gene to external factors, in this case, to the homologous IS.
It cannot be ruled out that the homologous IS, when acted on the cells at the invariable temperature of their cultivation, also induced some other serotypes. It is to be borne in mind that, when studying the serotype polymorphism among the D. anser clones of different origin, there were many clones that showed no cross-reactions and had, therefore, different serotypes under identical conditions of their cultivation, both at 17 and at 25°C (Uspenskaya, 1988, see above). However, this assumption has not yet been verified.
In this connection, our data on the temperature transformation of D. anser clones possessing «hybrid» serotypes are interesting to mention. Such clones were obtained in F2 from crosses of two clones with clearly distinct serotypes. When these clones were transferred from 25 to 17°C, their initial serotypes disappeared and were replaced by some new one (Uspenskaya and Yudin, 2001).
In the recent two decades, some evidence has been obtained for a multilevel regulation of serotypes in ciliates, specifically, at the levels of initiation of transcription and/or of stability of mRNAs (Gilley et al., 1980; Schmidt, 1988; Caron and Meyer, 1989; Stargell et al., 1990; Smith and Doerder, 1992; McMillan et al., 1993, 1995). The relative significance of these mechanisms, most likely, differs for different i-antigens and for different ciliate species (Love et al., 1988). One way to analyze this problem is to apply an inhibitor analysis to the serotype transformation by using inhibitors of transcription and/or translation. Thus, actinomycin D (AM-D) was used as an inhibitor of the serotype transformation (James, 1967; Preer, 1968; Sapra and Ammermann, 1973; Golinska and Bohatier, 1975; Sapra et al., 1976; Bohatier, 1977; Williams et al., 1985). At the same time, AM-D is known to cause a non-specific injury in ciliates and even to stimulate the serotype transformation (Austin et al., 1967; Sommerville, 1970).
For D. anser, the maximum sublethal concentration of AM-D was found to be 15 mg/ml; the cell divisions were suppressed almost completely by the first day after
the AM-D application, but the cell death was still insignificant. When the ciliates thus treated with AMD were transferred to the culture medium free of the inhibitor, the multiplication rate rapidly increased up to the normal. This indicates no serious non-specific injury of the ciliates. However, it was this sublethal AMD concentration that strongly affected the serotype transformation induced by the shift of temperature. We invariably observed a nearly total inhibition of the temperature transformation if AM-D was administered to the culture medium either a day prior to the temperature shift, or simultaneously, or even one day thereafter (Uspenskaya and Yudin, 1996). Judging from the immobilization, the ciliates retained their «old», or initial, serotype in the presence of AM-D, and a «novel» serotype did not appear. Hence, AM-D acted as an inhibitor of the serotype transformation, which, consequently, depended upon the RNA synthesis.
Even after a relatively long incubation with AM-D (4 to 5 days), the treated ciliates, when transferred to the antibiotic-free medium, started the serotype transformation as if anew, and the process failed to be completed by the time it was completed in the control cultures. However, a long observation revealed the process to proceed to completion, and no AM-D aftereffect was noticed. If AM-D was added to the culture medium at a later time (two days after the temperature shift or even later), either partial or even complete serotype transformation occurred after a time usually required for the full change of serotypes in the control cultures. These data suggest that RNA synthesis is necessary only at early stages of transformation, while later, the process is regulated at the post-transcriptional level (Uspenskaya and Yudin, 1996).
It was interesting to find out if AM-D inhibited the serotype transformation induced by the homologous IS as well. No change in the serotype (even transient one) was found if AM-D was introduced to the culture medium a day prior to the treatment of ciliates with the inducing IS, as well as on the first day after the treatment. Here, the effect of AM-D was, therefore, very similar to that in the case of the serotype transformation induced by the temperature change (Uspenskaya and Yudin, 1998b). This suggests that the serotype transformation induced by the homologous IS requires normal RNA synthesis at early stages of the process, similarly to the temperature transformation.
Thus, our study of the serotype transformation in D. anser has revealed that the replacement of one serotype with another takes a comparatively long time to be completed (usually, 5-6 days). This, particularly, might be convenient for investigating dynamics of the process under usual conditions or under the influence of various factors. As a result of the transformation,
some novel serotypes can be revealed among the admissible ones in a given clone.
In experiments on paramecia, the temperature transformation turned out to be an important aiding means in hybridological analysis of their serotypes (Beale, 1954). When we crossed the dilepti of different serotypes, it remained unclear whether the serotypes of the «parental» B and D clones were controlled by different alleles of the same genetic locus or by different loci: no segregation was observed among the F2 clones and all these clones had the «hybrid» phenotype -presumably due to some epigenetic alterations caused by crossing. We assumed that, if the maintenance of these epigenetic modifications was closely associated with the expression of the «hybrid» serotype, the temperature transformation of the F2 clones (i.e., temporary switching their «hybrid» serotype off) might have resulted in reversion of the corresponding epigenetic system to its initial state and in visualization of the proper genotypic segregation. Twenty five and twenty six F2 clones of D. anser obtained from back-crosses Fj x B and Fj x D, correspondingly, were tested once again for their serotypes. Each of them responded, more or less equally, to both ISs that were raised against the «parental» clones B and D. Therefore, all F2 clones had the «hybrid» phenotypes that were maintained during their asexual reproduction. The serotype transformation was induced in these clones by their transition from 25°C to 17°C. About two weeks after the completion of transformation, the clones were transferred back to 25°C and were repeatedly tested 7 and 20 days later with the same ISs (the anti-B and anti-D IS). As before, every clone (without exception) turned out to be immobilized with both ISs and, hence, demonstrated the «hybrid» phenotype, or the simultaneous expression of both «parental» i-antigens, despite the fact that the expression of these i-antigens was earlier switched off for two weeks by the temperature transformation. In a number of clones, different responses to two testing ISs were observed. There were some clones, whose reactions were the same before and after the transient change of the serotype. In some clones the reaction was changed after the transformation (Uspenskaya and Yudin, 2001).
In some models suggested to account for regulation of the differential i-antigens expression in Paramecium and Tetrahymena, the regulatory functions were ascribed to i-antigens themselves. Thus, an extensive experimentation on regulation of the surface antigens in Paramecium primaurelia allowed Capdeville (1979) to suggest that an i-antigen influenced positively its own synthesis, acting as the receptor of the external signals that determine the serotype alteration. Finger et al. (1995, 1995/1996) found the i-antigen under expres-
sion to form a complex with a specific 70 kDa protein; this protein revealed by the authors was free of the antigen inside the cell and inhibited transcription of the gene that codes the i-antigen. Earlier models of the kind were critically reviewed by Doerder (1979). In our opinion, the restoration of the «hybrid» phenotype (i.e., the reestablishment of the disturbed pattern of expression of corresponding genes) in all F2 clones of dilepti is inconsistent with the above suggestion of immediate participation of i-antigens in regulation of their expression.
When studying the serotypes in D. anser, we observed several cases of violating the principle of «mutual exclusion». It was already described that one of the D. anser clones simultaneously expressed two i-antigens over a long period of time. This was observed at temperatures that were intermediate between those inducing the complete transformation of one antigenic type into another. The absence of phenotypic segregation in the second hybrid generation of D. anser might be considered as another case of violating the normal mechanism of mutual exclusion of i-antigens. In most cases, however, only one type of the surface antigen is expressed in dilepti at a given time and under given circumstances; this rule of «mutual exclusion» indicates epigenetic component in the control of the serotypes in D. anser.
Perhaps, the only case of deviation from the rule of «mutual exclusion» between the i-antigens controlled by different (non-allelic) genes is reported in Paramecium. Under some special conditions, the individual lines of P. tetraurelia, stock 172, which expressed the surface antigen D, also started to express the antigen M (Margolin, 1956). In the author’s opinion, it was a certain state of long-lasting (permanent) transformation from one antigen to the other, and vice versa.
To summarize, the data discussed in this review and obtained from studying the lower ciliate Dileptus anser show that many features of the serotype system in this ciliate are similar to those described for the serotype systems of several classical species, the higher ciliates Paramecium and Tetrahymena. Particularly, in dilepti, some phenomena are observed which are usually described in paramecia and tetrahymenas in terms of epigenetic variation and inheritance. The study of the serotypes in Dileptus anser still is at the very initial stage, and dilepti are yet to become a popular laboratory model. Obviously, further study of serotypic diversity in dilepti, as well as further accumulation of knowledge on sexual process in this species, are certainly necessary. Different i-antigens are to be characterized biochemically. Poor knowledge of these biological features of the species strongly handicaps interpretation of experimental data.
The first attempt at hybridological analysis of the Dileptus serotypes was undertaken, although it did not comply with standards of the classical genetic analysis in many respects. Meanwhile, further studies of the serotype system in the lower ciliate Dileptus anser seem very promising and may be of obvious genetical interest.
Acknowledgments
The work was supported by the Russian Foundation for Basic Research (grant No 98-04-49683). The authors are grateful to Dr. L.Z. Pevzner for editing the English version of the manuscript.
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Address for correspondence: A.L.Yudin. Laboratory of Cytology of Unicellular Organisms, Institute of Cytology, Russian Academy of Sciences, Tikhoretsky Ave. 4, St. Petersburg, 194064, Russia. E-mail: yudin@cit.ras.spb.ru
