BiochemicalGenetics, Vol. 16, Nos. 9/10, 1978
Species Identification in Protozoa: Glucosephosphate Isomerase Variation in the Paramecium aurelia Group A. Tait 1
Received21 Dec. 1977--Final15Mar. 1978
Results are presented for intra- and interspecies variation in electrophoretic mobility of the enzyme glucosephosphate isomerase in the Paramecium aurelia species complex. Three new observations have been made." (1) the hitherto indistinguishable species 1 and 5 can be distinguished on the basis of GPI electrophoretic mobility, (2) the degree of intraspecies variation is much higher for GPI than for the previously studied mitochondrial dehydrogenases and esterases, and (3) several of the enzymatic variants observed in one species are apparently indistinguishable from some found in other species. The intraspecies variants found have been shown to be allelic, and, on the basis of the enzyme patterns of the heterozygotes, it is proposed that GPI is a dimeric enzyme determined by two loci. In view of the use of enzyme variation as a means of species identification in protozoa, these results suggest that the use of such methods can lead to underestimating the number of species and possibly to misclassification. The implications of these findings together with the results obtained with Tetrahymena are discussed. KEY WORDS:
Paramecium;glucosephosphateisomerase; species identification; enzymevaria-
tion. INTRODUCTION In the past 7 years a considerable amount of work has been done on species identification of protozoa by means of biochemical techniques rather than by the classical methods based on morphology, mating reactions, and genetic exchange. Starch gel electrophoresis of enzymes has been used both with This work was supported by a grant from the Science Research Council. Institute of Animal Genetics, Edinburgh, Scotland. 945 0006-2928/78/1000-0945505.00/0 © 1978Plenum Publishing Corporation
946
Tait
free-living protozoa (Tait, 1970a; Allen and Gibson, 1971; Borden et al., 1973) and parasitic protozoa (Carter, 1973; Carter and Walliker0 1975; Bagster and Parr, 1973; Godfrey and Kilgour, 1976; Gardener et al., 1974; Shirley, 1975). With morphologically similar protozoa comprising only asexual forms, difficult problems of species identification arise. The main question is: how different do two isolates need to be in terms of variation in electrophoretic enzyme mobility, to justify considering them as members of separate species, rather than variant forms of a single species? This question has been discussed by Nanney and McCoy (1976) with the aid of data from the Tetrahymena pyriformis group of species. These authors consider that isolates of a given species must be alike in at least 67% of electrophoretic enzyme characteristics, although most members of a species are much more similar than that. In this article some new results with the Paramecium aurelia group will be presented. The new observations involve variation of the enzyme glucosephosphate isomerase (GPI), which will be discussed in relation to the previously obtained results with other enzymes. The species "group" P. aurelia, as it has been called by Sonneborn (1975), offers particularly favorable material for this type of study. This complex comprises 14 syngens or species which are morphologically very similar but which can be precisely classified by their mating characteristics. Unlike Tetrahymena, which contains a considerable proportion of nonmating strains, all isolates of P. aurelia can be classified if sufficient work is done. By determining the amount of electrophoretic variation both within and between species (which have been previously classified), it is hoped to lay down some ground rules for distinguishing protozoan "species" solely by biochemical methods. Such rules could then be applied to organisms which do not mate. Previous work on intra- and interspecies enzyme variation in the P. aurelia group (Tait, 1970a; Allen and Gibson, 1971) has shown that all but one pair of the 14 species can be unambiguously distinguished by observations on enzymes controlled by eight loci (ICDs, ICDM, HBD, FUM, GDH, EstA, EstB, EstC). Although intraspecific variation has been found at six of these loci (Tait, 1968, 1970b; Allen and Golembiewski, 1972; Cavill and Gibson, 1972), the variant forms observed were unique and did not coincide with those observed in interspecies comparisons. Furthermore, the intraspecies variants were rare (occurring in less than 1% of stocks) in all species except species 2, although heat stability studies of the esterases in other species (Allen and Gibson, 1975) suggest that the low frequency of polymorphism may be more apparent than real. In the work to be described here on glucosephosphate isomerase (GPI), it will be shown that a high degree of polymorphism occurs and the variants within one species are often indistinguishable from variants in other species. These findings raise questions which may complicate the work of species
Species Identification in Protozoa
947
identification in nonmating protozoa, and this will be discussed later in this article. MATERIALS AND M E T H O D S
Stocks of P. aurelia from the 14 species used in this study were cultured in bacterized grass infusion. Previously the 14 species were referred to as varieties or syngens, but recently (Sonneborn, 1975) they have been designated as species and renamed as P. primaurelia, P. biaurelia, etc. For ease of comparison with earlier reports, the organisms will be referred to here as species 1,2 . . . . . 14. The methods for growth and concentration of cells together with preparation of cell extracts were as previously described (Tait, 1970a). Crosses and the isolation of Fl's and F2's from such crosses were performed as described by Sonneborn (1950). Cells were transferred to axenic medium by initial serial transfer in yeast extract and finally grown in axenic medium (Soldo et al., 1966). Electrophoresis of cell extracts was performed on 11% starch gel in 0.01 n tris-HC1, p H 8.0 (0.05 M tris-HC1, p H 8.0, in the electrode tanks), at 18 V/cm for 3 hr. The temperature was maintained at 2 C. After electrophoresis the surface was overlayed with 40 ml of 0.1 M tris-HC1, p H 8.0, containing 40 mg fructose-6-phosphate, 20 mg magnesium chloride, 5 mg NADP, 5 mg MTTtetrazolium, and 0.5 mg phenazine methosulfate. The gel was then incubated at 32 C until stained bands appeared (30-60 rain). Klebsiella aerogenes was used as a food organism and was grown in nutrient broth. Extracts were prepared by homogenizing washed pellets of bacteria under identical conditions to those used for the preparation of Paramecium extracts. The stocks of the 14 species of the Paramecium aurelia complex were either taken from the Edinburgh collection or were kindly supplied from Professor T. M. Sonneborn's laboratory, and are listed in Table I. Hydrolyzed starch was obtained from Connaught Research Laboratories, and all other chemicals were of analytical grade.
RESULTS
As Paramecium is grown on media containing bacteria, it is essential to show that the bands of GPI activity observed on starch gel are due to enzymes produced by paramecia rather than bacteria. With this in mind, extracts of paramecia grown on bacterized and axenic media, and extracts of bacteria alone, were run on gels and stained for GPI activity. The results are shown in
948
Tait
Table I. Stocks of Paramecium Screened for Electrophoretic Variation in Glucosephosphate Isomerase a Species
Stocks
Distribution of stocks used
1
16, 26, 60, 129, 143,144, 147, 168, 175, 257, 33~5,513, 520, 521,540, Hun 7 30, 72, 179, 193, 206,234, 2 6 0 , 291,310,339,527, 562, 1 0 1 0 , Sed-53, Hu 1/1, Cr 3/1 13, 79,152; 186,275,283 29, 32, 146, 163,172, 230, 2 8 0 , 315, 316, CANB-2, 4, 6, 8 76, 87, 120, 123, 132, 190, 210, 236, 311 101,159a,265,284, 302,303, 309, 326 227,228, 253,325 130, 202, 218,224, 276,281, 229, 300,307, 330, 565 204, 317, 323, 338,503
U.S.A., France, Russia, Hungary, Britain, Japan, Mexico, Chile, Peru U.S.A., Britain, Italy, Norway, Germany, Japan, Chile, Lebanon U.S.A. U.S.A., Italy, Holland, Japan, Australia, Peru U.S.A., Australia
2 3 4 5 6 7 8 9 10 11 12 13
223 248 246, 251,270, 273, 274 209,238,321
14
328
U.S.A., India, Kenya, Thailand U.S.A. (Florida) U.S.A., Panama, Uganda Britain, France, Germany, Russia U.S.A. U.S.A. U.S.A. France, Mexico, Madagascar Australia
a The distribution gives the various countries from which the stocks originated. Fig. 1. It is clear that the m a j o r b a n d o f activity observed in extracts o f cells g r o w n o n bacterized m e d i u m is due to the paramecia and not the bacteria, since the same b a n d appears in gels from axenically g r o w n cells. A faint, fast-migrating b a n d o f activity observed in preparations o f paramecia from bacterized m e d i u m appears to originate from bacteria, but it should be noted that this b a n d and the other b a n d observed in bacterial extracts b o t h migrate m u c h farther than any of the bands o f activity reported in this paper. The various stocks listed in Table I were screened for electrophoretic variants of GP1, and the m o s t frequent allele in each species is shown in Fig. 2. In the case o f species 7, two forms are shown as they occur with equal frequency. The relative positions o f the various bands o f activity were determined by mixing extracts from two different species and examining the relative mobility o f the bands o f activity. Several o f the extracts show multiple bands, and the possible basis o f this is discussed later. O n the basis o f these results, species 1 and 5 can n o w be distinguished by
Species Identification
in P r o t o z o a
949
~22~.::3
r/////A
r/////J
Fig. 1. Effect of culture conditions on enzyme bands stained for glucosephosphate isomerase. 513-bact, extract of stock 513 of Paramecium grown on bacterized grass medium; 513 ax, extract of stock 513 of Paramecium grown on axenic medium; ORIGI N Bact, extract of Klebsiella aerogenes. Details of conditions and methods are given in Materials and Methods.
i
i 513
bact
i 513 ax
i
Bact
enzyme comparisons. Previously these species, which are closely similar in many respects, other than enzyme characteristics, could be separated only on the basis of mating reactions and study of interspecific hybrids (Sonneborn, 1975). The results with GPI allow one to distinguish nine groups of species: I, species 1 and 7; II, species 2; III, species 3; IV, species 4, 8, and 10; V, species 5; VI, species 6; VII, species 9; VIII, species 1 I, 13, and 14; IX, species 12. The species which cannot be distinguished using GPI can be distinguished with other enzymes, and thus it is now possible to distinguish all 14 of the known species of the P. aurelia group. rrHHA V/////] k/////~ ~////A V/////]
'ORIGIN
SPECIES
L
•
1
12Z2Z~
V/////JV/////a
r/////Ar;7///A
~'JTz'A
~:.':,~;:,.,
eTZ'Z~V/N//A
Fs:+~a~-:~V-Z-/-/-da
I
I
2
i
3
I
4
I
5
i
6
I
7
i
8
I
9
I
10
~
11
i
~2
I
13
14
Fig. 2. Electrophoretic comparison of the most c o m m o n alleles of glucosephosphate isomerase from the 14 species of Paramecium. The pattern for species 7 indicates that both a three-banded and a single-banded (crosshatched) form occur with equal frequency. Conditions for electrophoresis and enzyme assay are described in Materials and Methods.
950
Tait
Before the enzyme phenotype of any organism can be used to identify its species unambiguously, however, the degree and nature of intraspecies variation must be ascertained. To this end, the enzyme types of a selection of stocks from each species (Table I) were determined by electrophoresis and the variant forms of GPI are shown in fig. 3. Two observations are immediately apparent: (1) this enzyme is polymorphic in eight out of the 12 species from which more than a single stock was available for study, and (2) in several instances the variant forms in one species seem to be indistinguishable from one or other forms in a second species. From these observations, and from comparison of the bands produced by the most common alleles in each species, it is clear that species identification would be impossible using GPI alone. With this enzyme, only four groups of species which do not have similar variants can be distinguished: I, species 1, 3, 7, 11, 13, 14; II, species 2, 4, 8, 10, 12; III, species 5, 6; IV, species 9. The frequency of the variant alleles within a given species appears to be quite high, taking into consideration the small number of stocks screened, although species 1 is the only one in which examples of variant alleles in more than one stock are found. The only species showing no forms of GPI found in any other species is species 9. The data on intra- and interspecies variation in this article have been combined with data from other enzymes (Tait, 1970a; Allen and Gibson, 1975) with the object of determining whether analysis of enzyme electrophoretic mobility alone is sufficient to distinguish any species from any other, even taking into account the possible occurrence of intraspecies variants. To do this, stocks were chosen (from each species) to represent the most common enzyme allele. The variant alleles were left out of this comparison as it is very difficult to incorporate them into such a calculation without taking a biassed
!
ORIGIN STOCK SPECIES
i
i 513
i 168 1
t 257
i
I 152
i 79
3
i
i 87
i 132
5
i
i 302
L 101
i 265
6
i 326
L 22'7
I iI 253
7
~ 300
I 299
8
I
L 317
i 323
9
L
i
i
27/* 2Z,6 12
Fig. 3. Electrophoretic variants of glucosephosphate isomerase in species 1, 3, 5, 6, 7, 8, 9, and 12. The stock numbers indicate strains showing this particular enzyme type. The clear bands of activity indicate the most common allele.
Species Identification in Protozoa
951
sample of stocks. Each comparison yields a coefficient of identity, defined as the fraction of enzyme patterns which could not be distinguished (see Borden et al., 1973). The results of such an analysis are shown in Table II. These figures are based on the ten loci examined so far, namely ICDs, ICDM, FUM, SDH, GDH, HBD, EstA, EstB, EstC, and GPL It can be seen that most of the species show low coefficients of similarity and so could readily be distinguished on this basis. Two of the species comparisons yield high similarity coefficients, namely species 1 compared to species 5 (90~), and species 7 and species 14 (70%), both values being within the range of similarity coefficient observed for intraspecies variation. These values are also greater than the 67% similarity coefficient used by Borden et al. (1973) as the criterion for distinguishing the "phenosets" of the Tetrahymena species complex. Thus by using the degree of similarity between isolates, where no morphological or other criteria of species identification are available, the number of species would tend to be underestimated. Furthermore, the similarity coefficient does not take into account intraspecies variation and where this occurs further misclassification may arise. Many of the stocks show multiple bands of GPI activity after electrophoresis, and some of the variation between stocks concerns the type and Table II. Values for the Similarity Coefficient Between Species Obtained by c o m p a r i n g All Pairwise Combinations of Strains from Each Species of P a r a m e c i u m a Species Species
1
2
3
4
5
6
7
8
9
10
11
12
13
14
1 2 3 4 5 6 7 8 9 10 11 12 12 14
100 20 60 20 90 40 60 40 50 30 40 40 30 60
100 40 10 20 20 30 20 20 10 20 10 10 20
100 20 50 20 50 30 30 20 30 30 30 40
100 30 30 20 50 40 50 20 50 30 20
100 50 50 40 60 40 40 50 30 60
100 30 40 50 20 20 30 10 40
100 40 50 20 30 30 40 70
100 40 30 20 30 30 40
100 20 50 40 20 50
100 30 60 30 20
100 30 40 50
100 40 30
100 50
100
a The similarity coefficient is measured as the n u m b e r of similar electropfioretic forms divided by the total n u m b e r of enzyme forms compared. This is expressed as a percentage, and the figures shown are based on the comparison o f t e n loci. The representative strain chosen for each species is that showing the most frequent allele observed.
952
Tait
number of multiple bands. Two questions have been asked about the nature of the multiple banding: (1) is it genetically determined? and (2) what is its molecular basis? To examine these points, crosses were made between stocks of species 1 which were variants of the common allele (GPI-1); three crosses were made between stocks 513 (GPI-2) and 168 (GPI-1), between 513 (GPI-2) and 257 (GPI-3), and between 168 (GPI-1) and 257 (GPI-3). As shown in Fig. 4, GPI-2 gives a single band of activity whereas GPI-1 and GPI-3 give three bands. The heterozygous F~'s were isolated from these crosses and extracts from them were subjected to electrophoresis; the enzyme patterns obtained together with those of the homozygous parental stocks are shown in Fig. 4. In all three crosses the heterozygotes show an additive enzyme pattern; i.e., any of the bands common to two variants are increased in relative intensity while those that are different are decreased in intensity. This was confirmed by diluting the F~ and parental extracts and determining the point at which a particular band was no longer detected. The F ~'s obtained from these crosses were then passed through autogamy, and the enzyme phenotype of the F2 clones was determined by electrophoresis. In P. aurelia the process of autogamy results in all loci becoming homozygous, and one would expect a 1 : 1 segregation of alleles on passing a population of heterozygotes through autogamy (for details, see Beale, 1954). Analysis of the F2's from two of the crosses showed a segregation of alleles (Table III) which was not significantly different from a 1:1 segregation of the two parental types. These results confirm that the variation observed within a species is controlled by allelic genes. The most likely basis for the multiple banding observed is that GPI is a dimer which is coded for by two structural loci. The pattern observed for GPI-1 and GPI-3 would be explained by the two subunits of the protein having a different charge, resulting in the production of a three-banded pattern. GPI-2 patterns would then be explained by mutation at one of these loci, producing in the case of GPI-2 two subunits with the same charge. These
+
ORIGIN STOCK
GPI type
=
L
I
=
i
513
F1
168
F1
2
2/1
1
1/3
I
257-
I
F1
3 3/2
I
513
2
Fig. 4. Electrophoretic pattern of glucosephosphate isomerase obtained from clones of heterozygotes for the three alleles identified in species 1.
Species Identification in Protozoa
953
Table III. Segregation of the Alleles GPI-1, GP1-2, and GPI-3 After Passing the Heterozygotes (a) GPI-1/GPI-2 and (b) GPI-I/GPI-3 T h r o u g h A u t o g a m y a Cross
GPI- I
GP1-2
GPI-3
Total
(a) (b)
16 14
27 --
-10
43 24
Z2 (a)=3.01 ~ )~z(b)=O.686J not significantat 5% level a Values of)~ 2 are given for the expected 1 : 1 segregation.
interpretations would also be consistent with the GPI pattern observed in heterozygotes. DISCUSSION The results presented in this article show that genetically determined electrophoretic variants of GPI occur at a relatively high frequency in many of the 14 species of P. aurelia, by comparison with the other enzymes previously studied. In previous studies on mitochondrial dehydrogenases (Tait, 1970a) and esterases (Allen and Gibson, 1975) a very low degree of polymorphism was observed in all species except species 2. A priori, three explanations seem possible: (1) too few electrophoretic conditions were used so that the degree of Variation was underestimated, (2) the intracellular location of the enzymes places restrictions on the degree of variation, and (3) the multimeric nature of GPI is different from that of the esterases and mitochondrial dehydrogenases. At present, we have insufficient data to decide conclusively whether any of these explanations are valid, but these possibilities could form the basis of further investigations. The main conclusion which can be drawn from the study of enzyme variation in P. aurelia is that the amount of intra- and interspecies variation which may be observed is very irregular and unpredictable, and depends on which enzymes are chosen for study as well as the number of isolates of a given species which are available. This should be borne in mind when using enzyme comparisons to identify "species" of nonsexual or sterile protozoa in the absence of other criteria. Although GPI distinguishes species 1 and 5 of P. aurelia, if mating analysis were not available it would not have been justifiable to separate these two species, since some stocks of species 1 differ from one another by more enzyme forms than are shown by. comparisons of species 1 and 5 stocks. Ideally, one requires a set of enzymes which are relatively invariant within species and clearly variant between species, but whether such
954
Tait
a set would be obtained in a random selection of enzymes is unpredictable. By examining the ten enzyme loci in P. aurelia, if mating analysis were not possible one would obtain an underestimate of the true number of species. In a parallel study of the Tetrahymena pyriformis complex, which unlike P. aurelia contains both mating and nonmating forms, Nanney and McCoy (1976) recommend that strains should be considered to belong to a given species (or phenoset) only if they are alike in at least 67% of enzyme mobilities, although most strains within a species showed a much higher degree of similarity. These conclusions were based on the study of eight enzymes. In Tetrahymena, polymorphism was not thought to confuse the separation of species, even though in the most thoroughly investigated one (syngen 1 or "T. thermophila") polymorphism was quite common. In conclusion, it should be stressed that although some problems arise from the use of enzyme variants as a means of identifying species in asexual protozoa, the method nevertheless has many advantages over others as regards precision and practicability. It is probably the most reliable method for separating the species of such protozoa as Trypanosoma and Plasmodium but should be used only when estimates of intraspecies variation have been made. ACKNOWLEDGMENTS I would like to thank Professor T. M. Sonneborn and members of his department for supplying a considerable number of stocks of various species and to thank Professor G. H. Beale for many helpful discussions.
REFERENCES Allen, S. L., and Gibson, I. (1971). Intersyngenic variations in the esterases of axenic stocks of Paramecium aurelia. Biochem. Genet. 5:161. Allen, S. L., and Gibson, I. (1975). Syngenic variations for enzymes of Paramecium aurelia. In Isozymes: Genetics and Evolution, Vol. IV, Academic Press, New York, p. 883. Allen, S. L. and Golembiewski, P. A. (1972). Inheritance of esterases A and B in syngen 2 of Paramecium aurelia. Genetics 71:469. Bagster, I. A., and Parr, C. W. (1973). Trypanosome identification by electrophoresis of soluble enzymes. Nature 244:364. Beale, G. H. (1954). In The Genetics of Paramecium aurefia, Cambridge University Press, Cambridge. Borden, D., Whitt, G. S., and Nanney, D. L. (1973). Electrophoretic characterisation of classical Tetrahymena pyriformis strains. J. ProtozooL 20:693. Carter, R. (1973). Enzyme variation in Plasmodium berghei and Plasmodium vinckei. Parasitology 66:297. Carter, R., and Walliker, D. (1975). New observations on the malaria parasites of rodents of the Central African Republic. Ann. Trop. Med. Parasitol. 69:187. Cavill, A., and Gibson, I. (1972). Genetic determination of esterases of syngens 1 and 8 in P. aurelia. Heredity 28:31.
Species Identification in Protozoa
955
Gardener, P. J., Chance, M. L., and Peters, W. (1974). Biochemical taxonomy of Leishmania. II. Electrophoretic variation of malate dehydrogenase. Ann. Trop. Med. Parasitol. 68:317. Godfrey, D. G., and Kilgour, V. (1976). Enzyme electrophoresis in characterizing the causative organism of Gambian trypanosomiasis. Trans. Roy. Soc. Trop. Med. Hyg. 70:219. Nanney, D. L., and McCoy, J. W. (1976). Characterization of the species of the Tetrahymena pyriformis complex. Tr. Am. Microsc, Soc, 95:664. Shirley, M. W. (1975). Enzyme variation in Eimeria species of the chicken. Parasitology 71:369. Soldo, A. T., Godoy, G. A., and van Wagtendonk, W. J. (1966). Growth of particle bearing and particle free Paramecium aurelia in axenic culture. J. Protozool. 13:492. Sonneborn, T. M. (1950). Methods in the general biology and genetics ofP. aurelia. J. Exp. Zool. 113:87. Sonneborn, T. M. (1975). The Paramecium aurelia complex of fourteen sibling species. Tr. Am. Microbiol. Soc. 94:155. Tait, A. (1968). Genetic control of/?-hydroxybutyrate dehydrogenase in P. aurelia. Nature 219:941, Tait, A. (1970a). Enzyme variation between syngens in P. aurelia. Biochem. Genet. 4:461. Tait, A. (1970b). Genetics of NADP-isocitrate dehydrogenase in P. aurelia. Nature 225:181.
Species Identification in Protozoa: Glucosephosphate Isomerase Variation in the Paramecium aurelia Group A. Tait 1
Received21 Dec. 1977--Final15Mar. 1978
Results are presented for intra- and interspecies variation in electrophoretic mobility of the enzyme glucosephosphate isomerase in the Paramecium aurelia species complex. Three new observations have been made." (1) the hitherto indistinguishable species 1 and 5 can be distinguished on the basis of GPI electrophoretic mobility, (2) the degree of intraspecies variation is much higher for GPI than for the previously studied mitochondrial dehydrogenases and esterases, and (3) several of the enzymatic variants observed in one species are apparently indistinguishable from some found in other species. The intraspecies variants found have been shown to be allelic, and, on the basis of the enzyme patterns of the heterozygotes, it is proposed that GPI is a dimeric enzyme determined by two loci. In view of the use of enzyme variation as a means of species identification in protozoa, these results suggest that the use of such methods can lead to underestimating the number of species and possibly to misclassification. The implications of these findings together with the results obtained with Tetrahymena are discussed. KEY WORDS:
Paramecium;glucosephosphateisomerase; species identification; enzymevaria-
tion. INTRODUCTION In the past 7 years a considerable amount of work has been done on species identification of protozoa by means of biochemical techniques rather than by the classical methods based on morphology, mating reactions, and genetic exchange. Starch gel electrophoresis of enzymes has been used both with This work was supported by a grant from the Science Research Council. Institute of Animal Genetics, Edinburgh, Scotland. 945 0006-2928/78/1000-0945505.00/0 © 1978Plenum Publishing Corporation
946
Tait
free-living protozoa (Tait, 1970a; Allen and Gibson, 1971; Borden et al., 1973) and parasitic protozoa (Carter, 1973; Carter and Walliker0 1975; Bagster and Parr, 1973; Godfrey and Kilgour, 1976; Gardener et al., 1974; Shirley, 1975). With morphologically similar protozoa comprising only asexual forms, difficult problems of species identification arise. The main question is: how different do two isolates need to be in terms of variation in electrophoretic enzyme mobility, to justify considering them as members of separate species, rather than variant forms of a single species? This question has been discussed by Nanney and McCoy (1976) with the aid of data from the Tetrahymena pyriformis group of species. These authors consider that isolates of a given species must be alike in at least 67% of electrophoretic enzyme characteristics, although most members of a species are much more similar than that. In this article some new results with the Paramecium aurelia group will be presented. The new observations involve variation of the enzyme glucosephosphate isomerase (GPI), which will be discussed in relation to the previously obtained results with other enzymes. The species "group" P. aurelia, as it has been called by Sonneborn (1975), offers particularly favorable material for this type of study. This complex comprises 14 syngens or species which are morphologically very similar but which can be precisely classified by their mating characteristics. Unlike Tetrahymena, which contains a considerable proportion of nonmating strains, all isolates of P. aurelia can be classified if sufficient work is done. By determining the amount of electrophoretic variation both within and between species (which have been previously classified), it is hoped to lay down some ground rules for distinguishing protozoan "species" solely by biochemical methods. Such rules could then be applied to organisms which do not mate. Previous work on intra- and interspecies enzyme variation in the P. aurelia group (Tait, 1970a; Allen and Gibson, 1971) has shown that all but one pair of the 14 species can be unambiguously distinguished by observations on enzymes controlled by eight loci (ICDs, ICDM, HBD, FUM, GDH, EstA, EstB, EstC). Although intraspecific variation has been found at six of these loci (Tait, 1968, 1970b; Allen and Golembiewski, 1972; Cavill and Gibson, 1972), the variant forms observed were unique and did not coincide with those observed in interspecies comparisons. Furthermore, the intraspecies variants were rare (occurring in less than 1% of stocks) in all species except species 2, although heat stability studies of the esterases in other species (Allen and Gibson, 1975) suggest that the low frequency of polymorphism may be more apparent than real. In the work to be described here on glucosephosphate isomerase (GPI), it will be shown that a high degree of polymorphism occurs and the variants within one species are often indistinguishable from variants in other species. These findings raise questions which may complicate the work of species
Species Identification in Protozoa
947
identification in nonmating protozoa, and this will be discussed later in this article. MATERIALS AND M E T H O D S
Stocks of P. aurelia from the 14 species used in this study were cultured in bacterized grass infusion. Previously the 14 species were referred to as varieties or syngens, but recently (Sonneborn, 1975) they have been designated as species and renamed as P. primaurelia, P. biaurelia, etc. For ease of comparison with earlier reports, the organisms will be referred to here as species 1,2 . . . . . 14. The methods for growth and concentration of cells together with preparation of cell extracts were as previously described (Tait, 1970a). Crosses and the isolation of Fl's and F2's from such crosses were performed as described by Sonneborn (1950). Cells were transferred to axenic medium by initial serial transfer in yeast extract and finally grown in axenic medium (Soldo et al., 1966). Electrophoresis of cell extracts was performed on 11% starch gel in 0.01 n tris-HC1, p H 8.0 (0.05 M tris-HC1, p H 8.0, in the electrode tanks), at 18 V/cm for 3 hr. The temperature was maintained at 2 C. After electrophoresis the surface was overlayed with 40 ml of 0.1 M tris-HC1, p H 8.0, containing 40 mg fructose-6-phosphate, 20 mg magnesium chloride, 5 mg NADP, 5 mg MTTtetrazolium, and 0.5 mg phenazine methosulfate. The gel was then incubated at 32 C until stained bands appeared (30-60 rain). Klebsiella aerogenes was used as a food organism and was grown in nutrient broth. Extracts were prepared by homogenizing washed pellets of bacteria under identical conditions to those used for the preparation of Paramecium extracts. The stocks of the 14 species of the Paramecium aurelia complex were either taken from the Edinburgh collection or were kindly supplied from Professor T. M. Sonneborn's laboratory, and are listed in Table I. Hydrolyzed starch was obtained from Connaught Research Laboratories, and all other chemicals were of analytical grade.
RESULTS
As Paramecium is grown on media containing bacteria, it is essential to show that the bands of GPI activity observed on starch gel are due to enzymes produced by paramecia rather than bacteria. With this in mind, extracts of paramecia grown on bacterized and axenic media, and extracts of bacteria alone, were run on gels and stained for GPI activity. The results are shown in
948
Tait
Table I. Stocks of Paramecium Screened for Electrophoretic Variation in Glucosephosphate Isomerase a Species
Stocks
Distribution of stocks used
1
16, 26, 60, 129, 143,144, 147, 168, 175, 257, 33~5,513, 520, 521,540, Hun 7 30, 72, 179, 193, 206,234, 2 6 0 , 291,310,339,527, 562, 1 0 1 0 , Sed-53, Hu 1/1, Cr 3/1 13, 79,152; 186,275,283 29, 32, 146, 163,172, 230, 2 8 0 , 315, 316, CANB-2, 4, 6, 8 76, 87, 120, 123, 132, 190, 210, 236, 311 101,159a,265,284, 302,303, 309, 326 227,228, 253,325 130, 202, 218,224, 276,281, 229, 300,307, 330, 565 204, 317, 323, 338,503
U.S.A., France, Russia, Hungary, Britain, Japan, Mexico, Chile, Peru U.S.A., Britain, Italy, Norway, Germany, Japan, Chile, Lebanon U.S.A. U.S.A., Italy, Holland, Japan, Australia, Peru U.S.A., Australia
2 3 4 5 6 7 8 9 10 11 12 13
223 248 246, 251,270, 273, 274 209,238,321
14
328
U.S.A., India, Kenya, Thailand U.S.A. (Florida) U.S.A., Panama, Uganda Britain, France, Germany, Russia U.S.A. U.S.A. U.S.A. France, Mexico, Madagascar Australia
a The distribution gives the various countries from which the stocks originated. Fig. 1. It is clear that the m a j o r b a n d o f activity observed in extracts o f cells g r o w n o n bacterized m e d i u m is due to the paramecia and not the bacteria, since the same b a n d appears in gels from axenically g r o w n cells. A faint, fast-migrating b a n d o f activity observed in preparations o f paramecia from bacterized m e d i u m appears to originate from bacteria, but it should be noted that this b a n d and the other b a n d observed in bacterial extracts b o t h migrate m u c h farther than any of the bands o f activity reported in this paper. The various stocks listed in Table I were screened for electrophoretic variants of GP1, and the m o s t frequent allele in each species is shown in Fig. 2. In the case o f species 7, two forms are shown as they occur with equal frequency. The relative positions o f the various bands o f activity were determined by mixing extracts from two different species and examining the relative mobility o f the bands o f activity. Several o f the extracts show multiple bands, and the possible basis o f this is discussed later. O n the basis o f these results, species 1 and 5 can n o w be distinguished by
Species Identification
in P r o t o z o a
949
~22~.::3
r/////A
r/////J
Fig. 1. Effect of culture conditions on enzyme bands stained for glucosephosphate isomerase. 513-bact, extract of stock 513 of Paramecium grown on bacterized grass medium; 513 ax, extract of stock 513 of Paramecium grown on axenic medium; ORIGI N Bact, extract of Klebsiella aerogenes. Details of conditions and methods are given in Materials and Methods.
i
i 513
bact
i 513 ax
i
Bact
enzyme comparisons. Previously these species, which are closely similar in many respects, other than enzyme characteristics, could be separated only on the basis of mating reactions and study of interspecific hybrids (Sonneborn, 1975). The results with GPI allow one to distinguish nine groups of species: I, species 1 and 7; II, species 2; III, species 3; IV, species 4, 8, and 10; V, species 5; VI, species 6; VII, species 9; VIII, species 1 I, 13, and 14; IX, species 12. The species which cannot be distinguished using GPI can be distinguished with other enzymes, and thus it is now possible to distinguish all 14 of the known species of the P. aurelia group. rrHHA V/////] k/////~ ~////A V/////]
'ORIGIN
SPECIES
L
•
1
12Z2Z~
V/////JV/////a
r/////Ar;7///A
~'JTz'A
~:.':,~;:,.,
eTZ'Z~V/N//A
Fs:+~a~-:~V-Z-/-/-da
I
I
2
i
3
I
4
I
5
i
6
I
7
i
8
I
9
I
10
~
11
i
~2
I
13
14
Fig. 2. Electrophoretic comparison of the most c o m m o n alleles of glucosephosphate isomerase from the 14 species of Paramecium. The pattern for species 7 indicates that both a three-banded and a single-banded (crosshatched) form occur with equal frequency. Conditions for electrophoresis and enzyme assay are described in Materials and Methods.
950
Tait
Before the enzyme phenotype of any organism can be used to identify its species unambiguously, however, the degree and nature of intraspecies variation must be ascertained. To this end, the enzyme types of a selection of stocks from each species (Table I) were determined by electrophoresis and the variant forms of GPI are shown in fig. 3. Two observations are immediately apparent: (1) this enzyme is polymorphic in eight out of the 12 species from which more than a single stock was available for study, and (2) in several instances the variant forms in one species seem to be indistinguishable from one or other forms in a second species. From these observations, and from comparison of the bands produced by the most common alleles in each species, it is clear that species identification would be impossible using GPI alone. With this enzyme, only four groups of species which do not have similar variants can be distinguished: I, species 1, 3, 7, 11, 13, 14; II, species 2, 4, 8, 10, 12; III, species 5, 6; IV, species 9. The frequency of the variant alleles within a given species appears to be quite high, taking into consideration the small number of stocks screened, although species 1 is the only one in which examples of variant alleles in more than one stock are found. The only species showing no forms of GPI found in any other species is species 9. The data on intra- and interspecies variation in this article have been combined with data from other enzymes (Tait, 1970a; Allen and Gibson, 1975) with the object of determining whether analysis of enzyme electrophoretic mobility alone is sufficient to distinguish any species from any other, even taking into account the possible occurrence of intraspecies variants. To do this, stocks were chosen (from each species) to represent the most common enzyme allele. The variant alleles were left out of this comparison as it is very difficult to incorporate them into such a calculation without taking a biassed
!
ORIGIN STOCK SPECIES
i
i 513
i 168 1
t 257
i
I 152
i 79
3
i
i 87
i 132
5
i
i 302
L 101
i 265
6
i 326
L 22'7
I iI 253
7
~ 300
I 299
8
I
L 317
i 323
9
L
i
i
27/* 2Z,6 12
Fig. 3. Electrophoretic variants of glucosephosphate isomerase in species 1, 3, 5, 6, 7, 8, 9, and 12. The stock numbers indicate strains showing this particular enzyme type. The clear bands of activity indicate the most common allele.
Species Identification in Protozoa
951
sample of stocks. Each comparison yields a coefficient of identity, defined as the fraction of enzyme patterns which could not be distinguished (see Borden et al., 1973). The results of such an analysis are shown in Table II. These figures are based on the ten loci examined so far, namely ICDs, ICDM, FUM, SDH, GDH, HBD, EstA, EstB, EstC, and GPL It can be seen that most of the species show low coefficients of similarity and so could readily be distinguished on this basis. Two of the species comparisons yield high similarity coefficients, namely species 1 compared to species 5 (90~), and species 7 and species 14 (70%), both values being within the range of similarity coefficient observed for intraspecies variation. These values are also greater than the 67% similarity coefficient used by Borden et al. (1973) as the criterion for distinguishing the "phenosets" of the Tetrahymena species complex. Thus by using the degree of similarity between isolates, where no morphological or other criteria of species identification are available, the number of species would tend to be underestimated. Furthermore, the similarity coefficient does not take into account intraspecies variation and where this occurs further misclassification may arise. Many of the stocks show multiple bands of GPI activity after electrophoresis, and some of the variation between stocks concerns the type and Table II. Values for the Similarity Coefficient Between Species Obtained by c o m p a r i n g All Pairwise Combinations of Strains from Each Species of P a r a m e c i u m a Species Species
1
2
3
4
5
6
7
8
9
10
11
12
13
14
1 2 3 4 5 6 7 8 9 10 11 12 12 14
100 20 60 20 90 40 60 40 50 30 40 40 30 60
100 40 10 20 20 30 20 20 10 20 10 10 20
100 20 50 20 50 30 30 20 30 30 30 40
100 30 30 20 50 40 50 20 50 30 20
100 50 50 40 60 40 40 50 30 60
100 30 40 50 20 20 30 10 40
100 40 50 20 30 30 40 70
100 40 30 20 30 30 40
100 20 50 40 20 50
100 30 60 30 20
100 30 40 50
100 40 30
100 50
100
a The similarity coefficient is measured as the n u m b e r of similar electropfioretic forms divided by the total n u m b e r of enzyme forms compared. This is expressed as a percentage, and the figures shown are based on the comparison o f t e n loci. The representative strain chosen for each species is that showing the most frequent allele observed.
952
Tait
number of multiple bands. Two questions have been asked about the nature of the multiple banding: (1) is it genetically determined? and (2) what is its molecular basis? To examine these points, crosses were made between stocks of species 1 which were variants of the common allele (GPI-1); three crosses were made between stocks 513 (GPI-2) and 168 (GPI-1), between 513 (GPI-2) and 257 (GPI-3), and between 168 (GPI-1) and 257 (GPI-3). As shown in Fig. 4, GPI-2 gives a single band of activity whereas GPI-1 and GPI-3 give three bands. The heterozygous F~'s were isolated from these crosses and extracts from them were subjected to electrophoresis; the enzyme patterns obtained together with those of the homozygous parental stocks are shown in Fig. 4. In all three crosses the heterozygotes show an additive enzyme pattern; i.e., any of the bands common to two variants are increased in relative intensity while those that are different are decreased in intensity. This was confirmed by diluting the F~ and parental extracts and determining the point at which a particular band was no longer detected. The F ~'s obtained from these crosses were then passed through autogamy, and the enzyme phenotype of the F2 clones was determined by electrophoresis. In P. aurelia the process of autogamy results in all loci becoming homozygous, and one would expect a 1 : 1 segregation of alleles on passing a population of heterozygotes through autogamy (for details, see Beale, 1954). Analysis of the F2's from two of the crosses showed a segregation of alleles (Table III) which was not significantly different from a 1:1 segregation of the two parental types. These results confirm that the variation observed within a species is controlled by allelic genes. The most likely basis for the multiple banding observed is that GPI is a dimer which is coded for by two structural loci. The pattern observed for GPI-1 and GPI-3 would be explained by the two subunits of the protein having a different charge, resulting in the production of a three-banded pattern. GPI-2 patterns would then be explained by mutation at one of these loci, producing in the case of GPI-2 two subunits with the same charge. These
+
ORIGIN STOCK
GPI type
=
L
I
=
i
513
F1
168
F1
2
2/1
1
1/3
I
257-
I
F1
3 3/2
I
513
2
Fig. 4. Electrophoretic pattern of glucosephosphate isomerase obtained from clones of heterozygotes for the three alleles identified in species 1.
Species Identification in Protozoa
953
Table III. Segregation of the Alleles GPI-1, GP1-2, and GPI-3 After Passing the Heterozygotes (a) GPI-1/GPI-2 and (b) GPI-I/GPI-3 T h r o u g h A u t o g a m y a Cross
GPI- I
GP1-2
GPI-3
Total
(a) (b)
16 14
27 --
-10
43 24
Z2 (a)=3.01 ~ )~z(b)=O.686J not significantat 5% level a Values of)~ 2 are given for the expected 1 : 1 segregation.
interpretations would also be consistent with the GPI pattern observed in heterozygotes. DISCUSSION The results presented in this article show that genetically determined electrophoretic variants of GPI occur at a relatively high frequency in many of the 14 species of P. aurelia, by comparison with the other enzymes previously studied. In previous studies on mitochondrial dehydrogenases (Tait, 1970a) and esterases (Allen and Gibson, 1975) a very low degree of polymorphism was observed in all species except species 2. A priori, three explanations seem possible: (1) too few electrophoretic conditions were used so that the degree of Variation was underestimated, (2) the intracellular location of the enzymes places restrictions on the degree of variation, and (3) the multimeric nature of GPI is different from that of the esterases and mitochondrial dehydrogenases. At present, we have insufficient data to decide conclusively whether any of these explanations are valid, but these possibilities could form the basis of further investigations. The main conclusion which can be drawn from the study of enzyme variation in P. aurelia is that the amount of intra- and interspecies variation which may be observed is very irregular and unpredictable, and depends on which enzymes are chosen for study as well as the number of isolates of a given species which are available. This should be borne in mind when using enzyme comparisons to identify "species" of nonsexual or sterile protozoa in the absence of other criteria. Although GPI distinguishes species 1 and 5 of P. aurelia, if mating analysis were not available it would not have been justifiable to separate these two species, since some stocks of species 1 differ from one another by more enzyme forms than are shown by. comparisons of species 1 and 5 stocks. Ideally, one requires a set of enzymes which are relatively invariant within species and clearly variant between species, but whether such
954
Tait
a set would be obtained in a random selection of enzymes is unpredictable. By examining the ten enzyme loci in P. aurelia, if mating analysis were not possible one would obtain an underestimate of the true number of species. In a parallel study of the Tetrahymena pyriformis complex, which unlike P. aurelia contains both mating and nonmating forms, Nanney and McCoy (1976) recommend that strains should be considered to belong to a given species (or phenoset) only if they are alike in at least 67% of enzyme mobilities, although most strains within a species showed a much higher degree of similarity. These conclusions were based on the study of eight enzymes. In Tetrahymena, polymorphism was not thought to confuse the separation of species, even though in the most thoroughly investigated one (syngen 1 or "T. thermophila") polymorphism was quite common. In conclusion, it should be stressed that although some problems arise from the use of enzyme variants as a means of identifying species in asexual protozoa, the method nevertheless has many advantages over others as regards precision and practicability. It is probably the most reliable method for separating the species of such protozoa as Trypanosoma and Plasmodium but should be used only when estimates of intraspecies variation have been made. ACKNOWLEDGMENTS I would like to thank Professor T. M. Sonneborn and members of his department for supplying a considerable number of stocks of various species and to thank Professor G. H. Beale for many helpful discussions.
REFERENCES Allen, S. L., and Gibson, I. (1971). Intersyngenic variations in the esterases of axenic stocks of Paramecium aurelia. Biochem. Genet. 5:161. Allen, S. L., and Gibson, I. (1975). Syngenic variations for enzymes of Paramecium aurelia. In Isozymes: Genetics and Evolution, Vol. IV, Academic Press, New York, p. 883. Allen, S. L. and Golembiewski, P. A. (1972). Inheritance of esterases A and B in syngen 2 of Paramecium aurelia. Genetics 71:469. Bagster, I. A., and Parr, C. W. (1973). Trypanosome identification by electrophoresis of soluble enzymes. Nature 244:364. Beale, G. H. (1954). In The Genetics of Paramecium aurefia, Cambridge University Press, Cambridge. Borden, D., Whitt, G. S., and Nanney, D. L. (1973). Electrophoretic characterisation of classical Tetrahymena pyriformis strains. J. ProtozooL 20:693. Carter, R. (1973). Enzyme variation in Plasmodium berghei and Plasmodium vinckei. Parasitology 66:297. Carter, R., and Walliker, D. (1975). New observations on the malaria parasites of rodents of the Central African Republic. Ann. Trop. Med. Parasitol. 69:187. Cavill, A., and Gibson, I. (1972). Genetic determination of esterases of syngens 1 and 8 in P. aurelia. Heredity 28:31.
Species Identification in Protozoa
955
Gardener, P. J., Chance, M. L., and Peters, W. (1974). Biochemical taxonomy of Leishmania. II. Electrophoretic variation of malate dehydrogenase. Ann. Trop. Med. Parasitol. 68:317. Godfrey, D. G., and Kilgour, V. (1976). Enzyme electrophoresis in characterizing the causative organism of Gambian trypanosomiasis. Trans. Roy. Soc. Trop. Med. Hyg. 70:219. Nanney, D. L., and McCoy, J. W. (1976). Characterization of the species of the Tetrahymena pyriformis complex. Tr. Am. Microsc, Soc, 95:664. Shirley, M. W. (1975). Enzyme variation in Eimeria species of the chicken. Parasitology 71:369. Soldo, A. T., Godoy, G. A., and van Wagtendonk, W. J. (1966). Growth of particle bearing and particle free Paramecium aurelia in axenic culture. J. Protozool. 13:492. Sonneborn, T. M. (1950). Methods in the general biology and genetics ofP. aurelia. J. Exp. Zool. 113:87. Sonneborn, T. M. (1975). The Paramecium aurelia complex of fourteen sibling species. Tr. Am. Microbiol. Soc. 94:155. Tait, A. (1968). Genetic control of/?-hydroxybutyrate dehydrogenase in P. aurelia. Nature 219:941, Tait, A. (1970a). Enzyme variation between syngens in P. aurelia. Biochem. Genet. 4:461. Tait, A. (1970b). Genetics of NADP-isocitrate dehydrogenase in P. aurelia. Nature 225:181.
