Esterase Variations between the 14 Syngens of PARAMECIUM AURELIA under Axenic Growth

Under axenic growth all 14 syngens of Paramecium aurelia have 4 types of esterases. The three major types (A, B and cathodal C) vary independently in electrophoretic mobility among the syngens. Using these three esterases, stocks can be keyed to a syngen, except for the groupings 1-3-5 and 7-13. Using 5 other enzymes only syngens 1 and 5 cannot be distinguished. Most syngens differ from each other in 6 out of the 8 enzymes. An axenically-grown stock of Paramecium multimicronucleatum collected in Costa Rica has the same types of esterases as P. aurelia. Two of the types (A and C) are similar in mobility to those found in syngens 7 and 13, but its B esterase differs in mobility from all the known syngens of P. aurelia.     

Intersyngenic variations in the esterases of bacterized Paramecium aurelia

The esterase isozymes were surveyed in bacterized stocks representative of all 14 syngens of Paramecium aurelia by starch gel electrophoresis. The properties of substrate specificity and independent variation of particular isozymes permit the ordering of the differences observed among stocks. Differences can arise from several sources: bacterial variation, intrasyngenic variation, and intersyngenic variation. Bacterial esterases tend to be found in certain zonal areas (see Rowe et al., 1971) and produce minor stock differences, which are erratic in their distribution. Unlike the situation found in Tetrahymena pyriformis, major intrasyngenic variations are rare in P. aurelia except in syngen 2. This lack of intrasyngenic variation is significant in view of the wide differences in geographic origin and micronuclear chromosome numbers among stocks within a syngen. It suggests that certain esterase genotypes must be under stringent selection within a syngen. The lack of intrasyngenic variation permits assessment of intersyngenic relationships. Syngens differ in a complex way from each other, suggesting that several gene differences may be involved. The syngens can be classified on the basis of their esterases. Syngens which have been shown to be more closely related in terms of cross-mating, breeding systems, and other criteria tend to be more similar in their esterase isozymes. The isozyme technique confirms relationships previously suggested among syngens and offers the promise of eventual assessment of evolutionary distances among syngens. However, establishment of these relationships will be clearer in the absence of bacteria.Supported by a Research Grant, GM-15879, from the National Institute of General Medical Sciences, U.S. Public Health Service     

Intraspecies Variability in the Esterases and Acid Phosphatases of Paramecium jenningsi and Paramecium multimicronucleatum: Assignment of Unidentified Paramecia; Comparison with the P. aurelia Complex1

ABSTRACT. Enzyme electrophoresis was exploited to identify stocks of paramecia previously not identified to particular species. Stocks collected in India and one from Panama belong to Paramecium jenningsi, while others collected in Panama or in Brazil are assignable to syngen 2 of P. multimicronucleatum on the basis of similarity of their esterase and acid phosphatase phenotypes. Inclusion of these doubled the numbers of stocks available in the two species, thereby facilitating examination of intraspecies variation and comparison of particular features of intraspecies variation found for the P. aurelia complex. Variant stocks were observed in P. jenningsi and in syngens 2, 3, and 4 of P. multimicronucleatum. In some cases the variant lacked the enzyme; in others, a change in mobility of the enzyme occurred that resulted in an electrophoretic form similar to one common in another species. Unique phenotypes were displayed by the variants of syngen 2 in P. multimicronucleatum. Hypervariability for Esterase B was observed in this syngen, where, in addition, several subtypes were seen for three other esterases. Unique phenotypes and hypervariability were also noted in P. biaurelia. Clustered variations were observed in these species and in the P. aurelia species. Unlike the situation for members of the aurelia complex, where lack of geographical differentiation between stocks in the same species is a unique feature, some such differentiation does occur in P. multimicronucleatum-2. The frequency of variant stocks in P. jenningsi was similar to that observed in the aurelia sibling species. In contrast, a significantly higher frequency of variant stocks was found in syngens 2, 3, and 4 of P. multimicronucleatum.     

Intersyngenic variations in the esterases and acid phosphatases of Tetrahymena pyriformis

The esterase and acid phosphatase isozymes were surveyed in strains of syngens 2–12 under conditions found to be optimal for syngen 1. Both intersyngenic and intrasyngenic variations were found. Comparisons of the esterases suggest that homologous enzymes are present in certain syngens and that some ordering of the variations with respect to syngen differences is possible. The acid phosphatases are highly polymorphic in different strains even within a syngen, and the variations cannot be ordered with respect to syngen differences. These results are discussed in terms of other types of studies directed at assessing syngen relationships and in terms of the sources of variation. It was concluded that only characters less vulnerable to intraclonal variation will be capable of revealing syngen relationships.Supported by a Research Grant, GM-15879, from the National Institute of General Medical Sciences, U.S. Public Health Service.     

Comparison of the Esterases and Acid Phosphatases in Paramecium multimicronucleatum,Syngens 1–5, P. jenningsi,P. caudatum,and the P. aurelia Complex1

ABSTRACT. Forty-eight stocks in Paramecium jenningsi, syngens 1–5 of P. multimicronucleatum, P. caudatum, P. primaurelia, P. biaurelia, and P. tetraurelia were grown axenically and tested for their esterases and acid phosphatases using starch gel electrophoresis. The five esterases and the acid phosphatases previously characterized in species of the P. aurelia complex were also found in P. jenningsi, and three to four of the esterases and the acid phosphatases were found in the P. multimicronucleatum species complex and in P. caudatum. Additional subtypes were observed for each of the enzyme phenotypes in these new (though here unnamed) species of Paramecium. Two of the new acid phosphatase subtypes, which depart radically in mobility and in pattern, were found in syngen 3 of P. multimicronucleatum and in P. caudatum. Except for syngens 1 and 5 in P. multimicronucleatum, the degree of similarity between syngens 1, 5 and 2, 3, and 4 appears to be very low—perhaps even lower than that seen for species in the aurelia complex. More realistically, the syngens of P. multimicronucleatum should be considered as separate species although they are not here given separate taxonomic names. Limited sharing of subtypes occurred between species in different species complexes. This observation suggests that the molecular distances between species complexes may be even greater than between species within a complex.     

Interspecies Crosses in Paramecium aurelia (Syngen 4 by Syngen 8)*

Two syngens (biologic species) of Paramecium aurelia that appear to be closely related were crossed. Parental stocks carrying different homozygous recessive marker genes were utilized to identify true hybrids (those that had undergone cross-fertilization followed by normal nuclear processes). From these crosses of syngens 4 and 8, 32% of the conjugants survived but only 9% (27% of the survivors) were true hybrids. All 19 viable hybrids recovered were cytoplasmically descended from the syngen 8 parent; but the viable nonhybrids were cytoplasmically descended from either of the 2 parents and with equal frequency from each. Surprisingly, the hybrids could not be infected with the symbiont kappa, even though they should have carried a K gene donated by their syngen 4 parent which is necessary and sufficient to allow infection of the syngen 4 parent stocks. The hybrids required special care to cultivate and were sterile, thus indicating that they are at an evolutionary dead end. However, one type of nonhybrid clone produced by the intersyngenic conjugants was able to produce viable progeny. It is speculated that genetic elements of the cortex and in the cytoplasm (e.g. mitochondria) could be transferred intersyngenically via this type of nonhybrid clone.     

Enzyme variation between syngens in Paramecium aurelia

Five enzymes (succinic dehydrogenase isocitrate dehydrogenase, glutamate dehydrogenase, -hydroxybutyrate dehydrogenase, and fumarase) in the ciliate Paramecium aurelia have been examined by starch gel electrophoresis. Relatively few variants were found among stocks belonging to a given syngen and only in two out of the five enzymes studied. Comparison of stocks belonging to different syngens, however, revealed many enzyme differences, which did not resemble intrasyngen variants. By studying the electrophoretic patterns of the enzymes of the 14 described syngens of Paramecium aurelia, it was found that only two syngens (Nos. 1 and 5) were indistinguishable. It is concluded that the use of starch gel electrophoresis of suitable enzymes provides a method of assigning a stock of paramecia to its syngen. Such a method may, in some cases, be less laborious than the standard one of making test matings and would, of course, be available for organisms which show no mating reaction.This work was carried out during the tenure of an M.R.C. Postgraduate Research Scholarship, and forms part of the material for a Ph.D. thesis at the University of Edinburgh.     

The Axenic Culture of Strains of the Various Syngens of Paramecium aurelia*

SYNOPSIS Strains of the various syngens of Paramecium aurelia respond differently to the culture media developed for Strain 299 of syngen 8. Not only is there a variety of response between the syngens, but strains belonging to the same syngen respond differently to the 3 growth media.     

The effects of growth conditions on the esterases of Paramecium aurelia

The esterase isozymes of Paramecium aurelia grown in the presence and absence of bacteria were examined by starch gel electrophoresis. Where present, bacteria make a positive contribution to the esterase zymograms. Different bacteria contribute different esterases. Paramecia growing in axenic medium possess a different esterase which is not produced by the same parmecia growing in the presence of bacteria. This axenic esterase disappears when bacteria are added back to the medium.Supported by a research grant from the Science Research Council.     

The Purification of DNA from the Genomes of Paramecium aurelia and Tetrahymena pyriformis1

SYNOPSIS. Methods are described for the recovery of highly purified DNA from Paramecium aurelia and Tetrahymena pyriformis in high yields. Our DNA is only slightly contaminated with RNA and carbohydrate, and little or no protein can be detected. We could not reduce the RNA (orcinol-positive material) by further treatment (sephadex or hydroxyapatite chromatography and preparative CsCl gradients). At the extreme our DNA is contaminated with 15–20% RNA but the real value is most likely considerably lower than this. The DNA we have prepared from Paramecium and Tetrahymena shows all the properties of double-stranded, high molecular weight DNA when characterized by temperature melting, CsCl density gradient centrifugation and hydroxyapatite and sephadex chromatography. When denatured, it absorbs to nitrocellulose filters. The 2 major results of importance from our work reported here are: (1) There is similarity in base composition of DNA from different syngens of Paramecium (28% G+C for syngens 1, 2, 4, 5, and 9 and 29–30% G+C for syngen 8) while there is variation between the syngens of Tetrahymena (24–31% G+C for syngens 1, 4, 7, 10, 11, and 12); (2) the density of any Paramecium DNA varies depending upon whether the cells are grown in the presence of bacteria or in axenic medium. Our results are compatible with observations previously reported for Tetrahymena but contradict those made for Paramecium. The earlier reports of differences in base composition between syngens of Paramecium are probably due in large part to the use of stocks grown on bacteria.     

The Syngens of Paramecium bursaria: New Mating Types and Intersyngenic Mating Reactions

SYNOPSIS. Three syngens of Paramecium bursaria have been identified amongst stocks collected in Scotland. These syngens probably correspond to the previously-described syngens 4, 5 and 6; they have been so named. In all 3 syngens 8 mating types have been found. An extensive series of intersyngenic mating reactions has been discovered between stocks of syngens 4 and 5, and between stocks of syngen 2 and syngens 4 and 5.     

Inter- and intrasyngenic variations of the hemoglobin components in Paramecium caudatum.

The ciliated protozoan Paramecium contains hemoglobin in heterogeneous monomeric forms. In particular, Paramecium caudatum is characterized by the presence of a major component called Hb10 and a basic component named bHb. We found that in P. caudatum both of these hemoglobin components show some variation according to stock. The types and distributions of these hemoglobin components were examined on 16 stocks in five different syngens and one stock in an unidentified syngen using high performance liquid chromatography. The results indicate that in a variety of stocks the major component, Hb10, was divided into three types, A, B or A + B, and that the basic hemoglobin component was composed of a combination of two or three variants out of four possible, i.e. bHb 1, bHb 2, bHb 3 and bHb 4. Neither the Hb10 types nor the bHb variants, however, could be used to distinguish syngen in P. caudatum, since all of the Hb10 types and bHb variants were widely distributed over syngens and identical profiles appeared to some stocks in different syngens.     

Mating Type Inheritance in Glaucoma*

SYNOPSIS. Mating was observed in collections of Glaucoma from 2 localities in Illinois. The collections could be divided into 2 syngens on the basis of mating type reactions. Corticotype analysis showed that syngen 1 was intermediate in meridian number between Glaucoma chattoni and Glaucoma scintillans, while syngen 2 had the same range as Glaucoma scintillans (Lee, unpublished). The major features of mating type inheritance in syngen 1 are that exconjugant clones usually have identical mating types and that mating types I and II appear in an approximately 1:1 ratio in successive generations. This is the result expected in genic mating type inheritance if one of the original parents was a homozygote and the other a heterozygote at the mating type locus. No significant immaturity period was found in these strains. Only 2 viable pairs were obtained from syngen 2 crosses, but results from these suggest that mating type inheritance may be epigenetic and a longer immaturity period may characterize this syngen.     

MOLECULAR DELINEATION OF SPECIES AND SYNGENS IN VOLVOCACEAN GREEN ALGAE (CHLOROPHYTA)1

Two species of the colonial green flagellate family Volvocaceae are worldwide in distribution yet exhibit contrasting species structure. Geographically disparate isolates of Gonium pectorale Mueller can interbreed while isolates of Pandorina morum Bory behave quite differently. More than 20 sexually isolated subpopulations occur within this species; these have been termed “syngens” (sensu Sonneborn). Because prezygotic barriers to mating cause intersyngen pairings to fail, breeding analyses cannot be used to estimate genetic relatedness among the syngens of P. morum. DNA comparisons provide an alternative method of assessing genetic relatedness. We compared the nucleotide sequence of the internal transcribed spacer (ITS) region of the nuclear ribosomal repeat among clones of P. morum and of G. pectorale. Members of syngens of P. morum with distribution restricted to one small geographical area show great similarity. Likewise, members of any syngen of worldwide distribution show near uniformity, even those from different continents. However, the ITS sequence of each syngen differs from that of other syngens. In contrast, G. pectorale, which has an ITS region that is remarkably uniform throughout the world, appears to consist of a single syngen within North America and Europe by mating tests. The molecular data are in complete conformity with previous syngen assignment. Because the latter is based on mating affinity, with two complementary mating types per syngen, the evolution of new mating type pairs appears to be the basis of microevolution in these algae. We infer that either P. morum is a more ancient species than G. pectorale or that P. morum has a less stable genome. In either case, the biogeographic distribution of certain syngens may reflect climatological changes of the past.     

Inter- and Intrasyngenic Variations of the Hemoglobin Components in Paramecium caudatum

ABSTRACT The ciliated protozoan Paramecium contains hemoglobin in heterogeneous monomeric forms. In particular, Paramecium caudatum is characterized by the presence of a major component called Hb10 and a basic component named bHb. We found that in P. caudatum both of these hemoglobin components show some variation according to stock. The types and distributions of these hemoglobin components were examined on 16 stocks in five different syngens and one stock in an unidentified syngen using high performance liquid chromatography. The results indicate that in a variety of stocks the major component, Hb10, was divided into three types, A, B or A + B, and that the basic hemoglobin component was composed of a combination of two or three variants out of four possible, i.e. bHb 1, bHb 2, bHb 3 and bHb 4. Neither the Hb10 types nor the bHb variants, however, could be used to distinguish syngen in P. caudatum , since all of the Hb10 types and bHb variants were widely distributed over syngens and identical profiles appeared to some stocks in different syngens.     

Enzyme Variation in Paramecium caudatum

Stocks of four “syngens” (syngens 1, 3, 12, 13) of Paramecium caudatum could not be distinguished on the basis of isozymic variations of six enzymes. Using the most common enzyme form as the standard for the syngen, we found a higher coefficient of identity between syngens (>90%) than within syngens (73%). These observations, combined with evidence for fertile matings among the groups, suggest that the groups do not warrant the status of syngens. True syngenic variation in P. caudatum is, however, clearly established on the basis of isozymic and breeding studies of wild stocks collected from various places. Some stocks from England have a close affinity with those from Japan, but stocks from Scotland and California must be placed in separate syngenic sets. Thus, P. caudatum is indeed a species complex, within which evolutionarily distinct groups (species = syngens) are identifiable. The definition of syngens on the basis of initial agglutination response is, however, unjustified.     

Genetic control of lactate dehydrogenase isozymes in Paramecium caudatum

The LDH isozymes were surveyed in bacterized cultures of syngens 1, 3, 12, and 13 of Paramecium caudatum by polyacrylamide gel electrophoresis. All the examined stocks of different syngens except one stock in syngen 3 had a single common LDH isozyme, and intra- and intersyngenic variation was not observed except for the one stock in syngen 3. Breeding data using the exceptional stock indicated that the LDH isozymes of P. caudatum are controlled by two codominant alleles at a single locus whose products aggregate randomly, forming a dimer.     

Method for the Simultaneous Establishment of Many Axenic Cultures of Paramecium*†

SYNOPSIS. A method is described for the simultaneous treatment of 42 (or more) stocks of Paramecium, and their adaptation to growth in axenic culture. Samples of dense cultures of these ciliates growing with Enterobacter aerogenes are rendered bacteria-free by migration through 2 sets of tubes containing Adaptation Medium (Peters' salts solution, stigmasterol, vitamins, and autoclaved E. aerogenes). The 2nd set of tubes contains Adaptation Medium plus antibiotics. Bacteria-free samples containing ～ 100 animals are then transferred to test tubes containing Adaptation Medium without antibiotics. This medium also serves as a growth medium. It supports indefinite growth of all Paramecium stocks tested. After adaptation to this medium, the ciliates can be grown in the axenic medium developed by Soldo, Godoy & van Wagtendonk. On a single trial at least half of the stocks can be expected to produce axenic cultures within 5 to 10 days by these procedures. The method has been applied successfully to several of the species of the Paramecium aurelia complex, to all syngens of Paramecium multimicronucleatum, to several stocks of Paramecium jenningsi, and to 1 stock each of Paramecium caudatum and Paramecium calkinsi. A modification of the method also works for Didinium nasutum.     

SEXUAL AND GENETIC ISOLATION IN THE COSMOPOLITAN ALGAL SPECIES PANDORINA MORUM

In an effort to analyze the nature of a taxonomic species in the microalgae, the major biological characters of various isolates of Pandorina morum have been studied. Seventy heterothallic clones from 31 collecting sites on three continents were found to represent 20 reproductively isolated groups, based on their ability to form zygotes when paired in all possible combinations. Successful germination of the zygotes and demonstrated fertility of their products suggest that each reproductively isolated group represents a syngen—a group of organisms sharing a common gene pool. A further indication of the close relationship of members of the same syngen is that its representatives all share a common chromosome number, time of division, and zygote distribution pattern, characters which vary between syngens. The known range of 18 of the syngens is less than 500 km. The other two syngens may have a world-wide distribution; they both have a haploid chromosome number of 5. Crosses between geographically isolated clones in these two syngens and one other give evidence of both pre- and postzygotic partial isolation within a syngen. Analysis of collections from two revisited sites demonstrated that representatives of a syngen can be recovered after 19 years have elapsed, and that a single pond may contain as many as four syngens. The results obtained for P. morum are compared with the information available on other microalgae with a similar breeding system, and the possible causes and consequences of the species structure are discussed.     

Growth of Particle-Bearing and Particle-Free Paramecium aurelia in Axenic Culture*

SYNOPSIS. Several strains of particle-bearing and particle-free Paramecium aurelia have been cultivated in an axenic medium composed of proteose peptone, trypticase, yeast nucleic acid, MgSO⁴.7H²O, TEM-4T (diacetyl tartaric acid esters of tallow monoglycerides), stigmasterol and a mixture of vitamins. The “yeast fraction,” an indispensable component of previous media used for the cultivation of these ciliates has been replaced by a mixture of trypticase, yeast nucleic acid and TEM-4T. Particle-bearing animals of stock 299 lambda, 138 mu, and 139 pi maintain their particles when cultivated in the medium, whereas particle-bearing animals of stock 51 kappa, 225 kappa and 114 signia do not. With the exception of stock 92 (syngen 3) the medium appears to be selective in its ability to support the growth of animals of the even- but not odd-numbered syngens of P. aurelia. Maintenance of the particles was dependent only to a small degree upon environmental conditions brought about by changes in pH and temperature. Division of the particles was found to be comparable with the division of the protozoan. Methods for the growth, maintenance and mass cultivation of particle-bearing P. aurelia are given in detail.