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.     

Esterase Variants in Four Species of the Paramecium aurelia Complex1

One hundred eighty-eight stocks of Paramecium primaurelia, P. biaurelia, P. tetraurelia, and P. octaurelia were grown axenically and tested for five esterases, visualized by starch gel electrophoresis, in a search for variant stocks. The five esterases can be distinguished on the bases of their substrate specificity, sensitivity to an inhibitor, and response to different growth conditions. This paper addresses the nature of the electrophoretic change in mobility of the variant stocks in order that species relationships can be more accurately assessed. Crosses carried out in all four species show that single genes determine the differences in mobility between variant and common subtypes. Extracts of variant stocks that gave similar patterns were run against each other, tested for their sensitivity to the inhibitor, and the pattern was compared to that found in extracts of stocks with variant and common subtypes in other species. The majority of the variants in P. primaurelia, P. tetraurelia, and P. octaurelia show an electrophoretic mobility characteristic of a common subtype, or a variant, in another species. The same proportion of variant subtypes as common subtypes have mobilities similar to esterase subtypes found in other species. Of the four species examined in this paper, P. tetraurelia and P. octaurelia appear to be most closely related on the basis of shared esterase subtypes. In P. biaurelia the mobilities of most of the variants are unique, as are the common esterase subtypes in this species. P. biaurelia stands out as having the greatest number of esterase subtypes, with very few of them homologous to subtypes found in other species. This observation supports the idea of greater diversification of stocks within P. biaurelia than for the other three species.     

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.     

Effect of Acetate on Esterase C Activity During the Growth Cycle of Paramecium*

SYNOPSIS Enhanced esterase C activity could be demonstrated by starch gel electrophoresis in various stocks of Paramecium spp. (P. primaurelia stocks 90 and 540, P. biaurelia stock 93, P. tetraurelia stock 29. P. pentaurelia stock 87, P. octaurelia stocks 31 and 300, and P. multimicronucleatum species 3, stock 8 MO) grown in Adaptation Medium. This esterase, however, was barely detectable when they were cultivated in Axenic Medium. Addition of trypticase to Adaptation Medium resulted in reduction of esterase C in the ciliates. This effect is ascribable to Na acetate present in trypticase. Since esterase C increased with the decrease in acetate concentration (as estimated by gas-liquid chromatography) during growth of Paramecium, acetate appears to be utilized by the cells. Sensitivity of esterase C to acetate occurs in all 6 species of Paramecium examined. Different stocks within a species may have different levels of sensitivity; in one case this is genetically determined. The results emphasize the importance of controlling and manipulating growth conditions for the assessment of inter- and intraspecies variations in the isozymes of Paramecium.     

Intersyngenic variations in the esterases of axenic stocks of Paramecium aurelia

The esterase isozymes were surveyed in axenic stocks of syngens 1, 2, 4, 5, 6, and 8 of Paramecium aurelia by starch gel electrophoresis. In paramecia there appear to be four types of esterases which are clearer in axenic than in bacterized stocks. Each type differs in its substrate specificity and/or its response to the inhibitor eserine sulfate. Minor variations in type D esterases sometimes occur in different extracts of the same stock and may result from changes in the temperature of growth of the cells or growth cycle differences. Differences in the mobility of the A, B, or C (cathodal) types of esterases may occur in different syngens. They also occur for the A and B types among stocks within a syngen, but the frequency is low, except in the case of syngen 2. Since each of the types of esterases varies independently, at least four and possibly more genes appear to specify the esterases in the species complex. Some pairs of syngens vary in their electrophoretic positions for all types of esterases. Other pairs have identical zymograms. This observation suggests that some syngens may differ from each other by as many as four esterase genes, while others may not differ at all. The difference between P. aurelia and Tetrahymena pyriformis in the degree of intrasyngenic variation observed for enzymes is discussed in relation to other types of characters, the organization of the genetic material in the macronucleus, the presence of symbionts, and their breeding systems. It is suggested that enzyme variation is achieved by the action of different selective forces in these two groups of ciliated protozoa.Supported by research grants from the National Institute of General Medical Sciences (GM-15879), U.S. Public Health Service, and from the British Medical Research Council.     

Interspecies Relationships in the Paramecium aurelia Complex: Acid Phosphatase Variation1

ABSTRACT. Up to five zones of acid phosphatase activity appear in gels after electrophoresis of detergent-treated extracts from 13 of the 14 species of the Paramecium aurelia complex. The overall pattern is somewhat similar for all species: differences in intensity and mobility of individual zones permit the grouping of these sibling species into eight groups. All 14 species can be identified using the procedure of enzyme electrophoresis, although two of them are more similar than is usually the case. Problems of misclassification are discussed in terms of the nature and frequency of variants. With the judicious choice of enzymes used to screen new stocks, these problems can be circumvented. Species relationships are updated using 11 enzymes. A dendrogram constructed from the matrix of genetic distances shows four clusters of species: (i) P. biaurelia, P. triaurelia; (ii) P. primaurelia, P. pentaurelia, P. sexaurelia, P. novaurelia; (iii) P. septaurelia, P. undecaurelia, P. tredecaurelia, P. quadecaurelia; and (iv) P. tetraurelia, P. octaurelia, P. decaurelia, P. dodecaurelia. Distances between the species are large, on the order of the differences between Drosophila species. The species are characterized by an extraordinary lack of geographical differentiation and great morphological similarity, which contrasts strongly with the molecular differentiation.     

Distribution of the Paramecium aurelia Species Complex (Protozoa,Ciliophora) in the Carpathian Chain of Poland*

Four species of the Paramecium aurelia complex were found in the Carpathian chain: P. primaurelia, P. biaurelia, P. tetraurelia and P. novaurelia. However, variation in the frequency of settlement of these species in the region was observed; P. biaurelia and P. novaurelia appeared with the greatest frequency, P. Primaurelia occurred less frequently. while P. tetraurelia was rather rare being found only in the Tatras.     

Genetic Analysis of Trichocyst Discharge of the Wild Stocks of Paramecium tetraurelia*

SYNOPSIS. Aberrant discharge of trichocysts in response to picric acid occurs in 8 of the 28 wild stocks of Paramecium tetraurelia. There are at least 4 distinguishable phenotypes: nondischarge, stocks 139, 163, 169, and 242; temperature-sensitive nondischarge, stock 126; leaky nondischarge, stock 203; and a clonally unstable phenotype, stocks 146 and 148. From each of these stocks a single recessive gene causing nondischarge has been isolated by backcrosses to stock 51. The original stocks 126, 146, and 148 possess other genes which affect the extracted genes. The copper resistance locus is ～ 10 centiMorgans from nd169 and nd242, but none of the other nondischarge genes are linked to 6 marker loci. The genes nd169 and nd242 are only 0.5 centiMorgans apart making them the closest known pair of loci in P. tetraurelia. The genes nd126 and nd242 are distinguishable alleles at the same locus and the genes nd146 and nd148 are apparently identical alleles. The large number of loci involved in producing a similar phenotype in different stocks supports the idea that mutation is much more important than gene flow in this highly inbreeding species.     

Seasonal and spatial variability of species occurrence of the <Emphasis Type="Italic">Paramecium aurelia</Emphasis> complex in a single natural pond (Protista,Ciliophora)

The geographic distribution and temporal occurrence of ciliates are still little known. In the present article, the occurrence of the Paramecium aurelia species complex in a natural pond situated in Kraków (Opatkowice) was investigated in different seasons in two following years. A sequence of species occurrence of the P. aurelia complex was observed. Always, paramecia were found only in some sampling points among six points sampled each time and not necessarily in the same ones. Paramecia appearing in one habitat (water body) might occupy different niches characterized by various environmental features suitable for paramecia. The following species were found in the pond: P. biaurelia, P. triaurelia, P. tetraurelia, P. pentaurelia, and P. dodecaurelia. The occurrence of some rare species (P. tetraurelia, P. pentaurelia, and P. dodecaurelia) may be connected with migrating birds which can transport paramecia with drops of water from other water bodies. If a species was observed in successive seasons or years, the possible genetic variation was investigated by analysis of sequences of LSU rDNA and mitochondrial cytb gene fragments. Among the studied species (P. biaurelia, P. triaurelia, P. pentaurelia, and P. dodecaurelia) only P. dodecaurelia showed haplotype variation in different seasons and sampling points, probably caused by the colonization of the pond by different populations of paramecia.     

Species identification in protozoa: Glucosephosphate isomerase variation in the Paramecium aurelia group

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.     

Worldwide sampling reveals low genetic variability in populations of the freshwater ciliate <Emphasis Type="Italic">Paramecium biaurelia</Emphasis> (<Emphasis Type="Italic">P. aurelia</Emphasis> species complex,Ciliophora, Protozoa)

Species (or cryptic species) identification in microbial eukaryotes often requires a combined morphological and molecular approach, and if possible, mating reaction tests that confirm, for example, that distant populations are in fact one species. We used P. biaurelia (one of the 15 cryptic species of the P. aurelia complex) collected worldwide from 92 sampling points over 62 years and analyzed with the three above mentioned approaches as a model for testing protistan biogeography hypotheses. Our results indicated that despite the large distance between them, most of the studied populations of P. biaurelia do not differ from each other (rDNA fragment), or differ only slightly (COI mtDNA fragment). These results could suggest that in the past, the predecessors of the present P. biaurelia population experienced a bottleneck event, and that its current distribution is the result of recent dispersal by natural or anthropogenic factors. Another possible explanation for the low level of genetic diversity despite the huge distances between the collecting sites could be a slow rate of mutation of the studied DNA fragments, as has been found in some other species of the P. aurelia complex. COI haplotypes determined from samples obtained during field research conducted in 2015–2016 in 28 locations/374 sampling points in southern Poland were shared with other, often distant P. biaurelia populations. In the Kraków area, we found 5 of the 11 currently known COI P. biaurelia haplotypes. In 5 of 7 reservoirs from which P. biaurelia was obtained, two different COI haplotypes were identified.     

Esterase D in Cebus apella from the Amazonian region*

Research programs involving the study of genetic variation of proteins have been carried out both in humans and catarrhine primates but rarely in New World species. Considering the great possibilities offered by protein variations as genetic markers in systematic and evolutionary studies in this group of organisms, the authors are specially interested in the investigation of genetic polymorphisms of blood proteins in Amazonian primates. In this paper we describe the electro phoretic patterns of erythrocyte esterases obtained from blood samples of 57 Cebus apella specimens. Blood hemolysates from Cebus display four main set of bands in azo-coupled stained gels. These bands are identified as esterases P, A1, A2 and B by their electro phoretic migration, substrate specificity and eserine reaction. The use of the fluoregenic reagent 4-methylumbelliferyl acetate revealed the presence of a fifth set of enzymes not detected by the azo-coupled staining method. This ser of enzymes, probably a polymorphic genetic system, was named ESD as in humans.     

Evolutionary divergence of mitochondrial DNA from Paramecium aurelia

Summary Mitochondrial (mt) DNA from four sibling species within the Paramecium aurelia complex, including stocks of different geographic origin and mutants, were analyzed using four 6-bp recognition site and one 4-bp recognition site endonucleases and the sequence divergence was estimated using Upholt's (1977) statistical procedure. All four species were readily distinguishable regardless of the restriction endonuclease employed. With intraspecies comparisons, no differences were observed which could be accounted for on the basis of geographic origin. Except for species 4, each stock (and mutant) gave a species-specific fragment pattern. For species 4, while the patterns were distinct from the other species, two species-specific type of patterns were found, designated A and B. The sequence divergence between these was estimated to be between 1 and 2 percent. With interspecies comparisons, the sequence divergence ranged from 3.9 to 10.3% with the greatest divergence being between species 1 and 4, and the least between species 1 and 5. The similarity between species 1 and 5 is in accord with other criteria for interspecies comparisons. The degree of sequence divergence measured here in Paramecium mt DNA is well within the range reported for rodents and primates. All four species mt DNA were cleaved to many DNA fragments by DPN II, an enzyme which recognizes non-methylated sites, and not by DPNI, the methyl-site specific counterpart of DPN II, suggesting that mt DNA from Paramecium aurelia is not appreciably methylated, if at all.     

Use of AFLP Analyses to Assess Genetic Variation in Morone and Thunnus Species

The amplified fragment length polymorphism (AFLP) technique was used to assess genetic variation across the genera of 2 Morone and 3 Thunnus species. The AFLP profiles from 23 primers revealed higher levels of polymorphism in each of 3 Thunnus species than in either of the 2 Morone species. However, extensive variation (20 of 23 primers) was observed between the 2 Morone species, but much less variation was observed among the 3 Thunnus species. In addition, comparisons of banding patterns indicated that albacore is divergent from both Atlantic northern bluefin tuna and yellowfin tuna. This result is consistent with the findings of several previous studies employing either allozymes or mitochondrial DNA. Overall, these results demonstrate that AFLP is a useful technique for the assessment of both intraspecies and interspecies variation of fish. Furthermore, the species-specific patterns produced by AFLP can be used for the identification of closely related species.     

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.     

A technique for identifying the roots of different species in mixed samples using nuclear ribosomal DNA

Abstract. Question: Is it possible to determine the species composition of root samples containing multiple species, without first disentangling individual roots? Methods: The internal transcribed spacer (ITS) region of nuclear ribosomal DNA was amplified and sequenced from four California annual grassland species (two Poaceae and two Asteraceae). Restriction enzymes that cut the ITS region of each species into uniquely sized fragments were identified based on DNA sequence variation of the ITS regions. Mixed root samples were analysed to test the ability of the method to identify the presence or absence of each species in multi‐species samples. Results: The technique successfully identified species present in multi‐species samples. ITS regions were shorter in Poaceae than in Asteraceae, so size differences alone were sufficient to distinguish these taxonomic groups. At the species level, digestion of ITS regions with the appropriate restriction enzymes yielded at least one uniquely sized fragment for each species. Conclusions: This method is the first to identify the species composition of mixed root samples. It should be applicable to most plant species because the ITS region is flanked by universal primers and most species have unique ITS sequences. The ability to determine species‐specific rooting distributions has broad applications in vegetation science.     

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     

Genetic linkage relationships of eight enzyme/electromorph loci in Anopheles minimus

Two laboratory stocks of Anopheles minimus, each fixed for variant electromorphs of esterases, aspartate aminotransferase, hydroxyacid dehydrogenase, phosphogluconate dehydrogenase, mannose phosphate isomerase and glycerol dehydrogenase were used to assess linkage relationships between presumed gene loci controlling this variation. The two F₁, which had been obtained from crossing the stocks, were backcrossed to a parental stock. Three loci controlled the esterases and one locus each of the other enzymes. Mpi is sex-linked. The rest are autosomal and suggested relationships are: Pgd 2.3% recombination from Aat and unlinked to any other loci; Est-1-33.8%-Est-3-31.5%-Est-2-21.0%-Had. Gcd is unlinked to any other locus.There was evidence of strong interaction between the X chromosome of one stock and autosomes of the other in which individuals bearing the X chromosome of the one suffered relatively greater mortality and had delayed development with respect to other genotypic classes.     

INSECTICIDE RESISTANCE IN THE GROUND SPIDER,Pardosa sumatrana (THORELL, 1890; ARANEAE: LYCOSIDAE)

Elevated levels of insecticides detoxifying enzymes, such as esterases, glutathione S‐transferases (GSTs), and cytochrome P‐450 monooxygenases, act in the resistance mechanisms in insects. In the present study, levels of these enzymes in the insecticide‐resistant ground spider Pardosa sumatrana (Thorell, 1890) were compared with a susceptible population (control) of the same species. Standard protocols were used for biochemical estimation of enzymes. The results showed significantly higher levels of nonspecific esterases and monooxygenases in resistant spiders compared to controls. The activity of GSTs was lower in the resistant spiders. Elevated levels of nonspecific esterases and monooxygenases suggest their role in metabolic resistance in P. sumatrana. The reduced levels of total protein contents revealed its possible consumption to meet energy demands.     

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.