Helmut J. Schmidt

Fast and Accurate Identification of European Species of the Paramecium aurelia Complex by RAPD-Fingerprints

A bstractCiliates of the genus Paramecium have a unique cortical pattern after silver staining, and can be easily identified as paramecia in any sample from small ponds or other freshwater collection areas. Furthermore, readily detectable morphological characteristics serve as guides to distinguish between species of paramecia by light microscopy of living cells. Alternatively different species of the P. aurelia complex appear morphologically identical, often despite striking differences in their ecology or life strategies. This has severely hampered ecological studies [field work]. There are only a few data sets available concerning the ecology of free living paramecia in natural habitats, because there is no feasible method for multiple determinations of Paramecium species that are often required during field studies. With the adaptation of the Random Amplified Polymorphic DNA–Polymerase Chain Reaction (RAPD–PCR) fingerprint technique, we succeeded in performing a fast and simple identification of European Paramecium aurelia species. We developed a 10-mer random primer that generated fingerprints highly specific for nine different P. aurelia species. The diagnostic band pattern was the same for different stocks of the same species, despite different geographic origins. This method has already been used successfully to identify some unknown clones collected from natural environments in southwestern Germany. This technique can be used for the identification of species and for studying elements of population structure. Field studies are now possible to answer basic questions concerning the ecology of Paramecium. These are necessary to develop a general model for limnological processes in freshwater ecosystems at a scale of a 100-μm organism.

A combination of genetics with inter- and intra-strain crosses and RAPD-fingerprints reveals different population structures within the Paramecium aurelia species complex

Summary A combination of classical methods with modern molecular biological techniques has been used to detect differences in geographical population structure between two species of the Paramecium aurelia complex. We combined interand intra-strain crosses via mating type reactions and random amplified polymorphic DNA-Polymerase chain reaction (RAPD-PCR). This RAPD-PCR method is quite favourable to identify sister species of the P. aurelia species complex and to detect different population specific genotypes within sister species. A comparison of the DNA fingerprints of several P. tri-aurelia strains as well as P. sexaurelia strains with the percentage of surviving hybrid strains of strain crosses in Fl and F2 generations revealed surprisingly results. A high percentage of offspring survive of Fl and F2 generations was typically observed in crosses with different P. triaurelia strains. The fingerprints distinguished three different genotypes within the investigated P. triaurelia strains. All three genotypes were distributed throughout the European regions where these strains were collected. P. sexaurelia, in contrast, showed low survival in crosses between regions and in some cases the stocks from different regions would not mate. Within P. sexaurelia four different fingerprint genotypes were found, but stocks from a single region had only a single genotype. We interpret these results as evidence for genetic gene flow and genetic isolation, respectively. The differences indicate two different life history strategies as a consequence of different degrees of inbreeding. P. triaurelia was confirmed as a moderate inbreeder and P. sexaurelia was discovered to be an extreme inbreeder, with breeding restricted to individuals belonging to the same genotype. We argue that not only different species complexes within the genus Paramecium have different life history and evolution strategies but remarkable ecogenetic differences are also true for sibling species within the same species complex.

Antigenic variation in ciliates : Antigen structure, function, expression

ABSTRACT. In the past decades, the major focus of antigen variation research has been on parasitic protists. However, antigenic variation occurs also in free‐living protists. The antigenic systems of the ciliates Paramecium and Tetrahymena have been studied for more than 100 yr. In spite of different life strategies and distant phylogenetic relationships of free‐living ciliates and parasitic protists, their antigenic systems have features in common, such as the presence of repeated protein motifs and multigene families. The function of variable surface antigens in free‐living ciliates is still unknown. Up to now no detailed monitoring of antigen expression in free‐living ciliates in natural habitats has been performed. Unlike stochastic switching in parasites, antigen expression in ciliates can be directed, e.g. by temperature, which holds great advantages for research on the expression mechanism. Regulated expression of surface antigens occurs in an exclusive way and the responsible mechanism is complex, involving both transcriptional and post‐transcriptional features. The involvement of homology‐dependent effects has been proposed several times but has not been proved yet.

Caedibacter caryophila sp. nov., a Killer Symbiont Inhabiting the Macronucleus of Paramecium caudatum

Caedibacter caryophila sp. nov. lives in the macronucleus of certain strains of Paramecium caudatum. The type strain is 221, carried in P. caudatum C221. C. caryophila is distinguished from other caedibacteria on the basis of host specificity, R body morphology, behavior of R bodies, and guanine-plus-cytosine content of its deoxyribonucleic acid.

ARDRA and RAPD-fingerprinting reject the sibling species concept for the ciliate Paramecium caudatum (Ciliophora, Protoctista)

Morphologically indistinguishable sibling species also known as syngens are a characteristic taxonomic feature of the ciliate genus Paramecium. This has been convincingly demonstrated for the P. aurelia species complex. For a long time this feature has also been assumed for P. caudatum. Classical morphology based techniques of taxonomic analysis are often inefficient to study sibling specie. We therefore investigated 14 P. caudatum strains of seven supposedly different syngens using random amplified polymorphic DNA (RAPD)‐fingerprinting and amplified ribosomal DNA restriction analyses (ARDRA, Riboprinting). The RAPD patterns revealed by five different random primers were similar between the different strains of the same syngen (similarity index ranging from 73 to 91%) and also between strains of supposedly different syngens (similarity index ranging from 67 to 91%). The amplified 18S rRNA‐fragments of supposedly different syngens, as well as the restriction patterns of these fragments digested by five different endonucleases, were identical for all investigated P. caudatum stains. Consequently we reject the sibling species hypothesis for P. caudatum. According to our molecular analysis, P. caudatum is not a species complex, but just one single species.

Rediscovery of Paramecium nephridiatum Gelei, 1925 and its Characteristics

Paramecium nephridiatum Gelei. 1925, was rediscovered. It is a euryhaline brackish‐water species that morphologically resembles Paramecium woodruffi. but with multiple contractile vacuole pores. The general morphology, morphometry. and random amplified polymorphic DNA (RAPD) fingerprint patterns are presented for a number of the stocks collected around the world.

Genetically controlled expression of surface variant antigens in free-living protozoa.

Besides parasitic protozoa some free-living ciliates have the ability to exhibit alternative types of proteins on their cell surface (Sonneborn, 1948; reviews: Schmidt, 1988; Bleyman, 1996; Schmidt, 1996). A range of these exchangeable structurally different surface proteins has been detected in species of the genera Paramecium(Ciliophora) andTetrahymena(Ciliophora). The presence of variable surface proteins is detected by immunochemical techniques. Injections of Parameciaor cells ofTetrahymenaspecies into mammals induce the production of antibodies against surface proteins of the injected cells. Therefore the proteins also are called surface antigens. Treatment of livingParameciaor of Tetrahymenacells with the homologous serum results in immobilization of the ciliates at low serum concentrations, or leads to cell death at higher concentrations. Because of the immobilizing effect of antibodies the corresponding surface antigens also are called “immobilization-antigens”, or “i-antigens.” Recent research involves the detection of mRNA specific for surface antigens by Reverse Transcriptase-PCR techniques (H.W. Breiner, H.J. Schmidt, J. Kusch, unpublished results ), with the aim to investigate single cells from field samples and a possible ecological function of these molecules. Cells that express the same type of surface antigen and therefore can be immobilized by the same antiserum belong to one serotype. Within genetically identical clones cells sometimes appear that are resistant against immobilization via antibodies. These cells induce the production of a different type of antibody after their injection into mammals. The cells represent a further serotype within the ciliate clone. In this way a range of serotypes, corresponding to different surface antigens, was observed in several species of Parameciumand of Tetrahymena(Nanney & Dubert, 1960; Koizumi, 1966; Hiwatashi, 1967; Juergensmeyer, 1969; Sonneborn, 1974; Steers & Barnett, 1982). Seven different serotypes are known forParamecium primaureliaand twelve for P. tetraurelia. Most of the known strains of these species cannot express all of the surface antigen types, e.g., types S, G and D only can be observed in most strains of P. primaurelia. Different surface antigens are generally mutually exclusive (Beale, 1957). Ciliates express only one type of surface antigen at constant environmental conditions, although genes for other surface antigens are present. A property of serotype expression is the ability to switch to another serotype. Among the stimuli for transformation are changes in temperature, pH, and the kind of growth medium, or UV radiation and proteolytic enzymes (Sonneborn, 1970). The inheritance of serotype genes follows Mendelian rules (Beale, 1957). The function of these surface proteins and the mechanism responsible for the variability of their expression are unknown. One suggestion is that they may be a buffer or defense against environmental biotic or abiotic factors (Preer, 1986). Paramecialacking variable surface proteins have never been found.

The Toxic Symbiont Caedibacter caryophila in the Cytoplasm of Paramecium novaurelia.

Endosymbiotic bacteria were observed to inhabit the cytoplasm of the freshwater ciliateParamecium novaurelia. Transmission electron microscopy and toxicity tests with sensitive paramecia showed that the endosymbionts belong to the genusCaedibacter. The bacteria conferred a killer trait to their host paramecia. The production of a proteinaceous inclusion body (“R-body”) in the bacterial cell makes them toxic to other paramecia after they become enclosed in food vacuoles. R-bodies ofCaedibacter sp were associated with phages, which are known in most otherCaedibacter species to code for the R-body proteins. The killer-effect ofP. novaurelia on sensitiveP. caudatum strains was of the “paralysis” type, which is a characteristic of the symbiont speciesCaedibacter caryophila. Until nowC. caryophila was known to inhabit the macronucleus ofParamecium caudatum only. Sequencing of the 16S rRNA-gene proved thatCaedibacter sp from the cytoplasm ofP. novaurelia belongs to the speciesC. caryophila as well. The rDNA-sequence of 1695 bp length differed in a total of only 1 bp from the corresponding gene inC. caryophila from the macronucleus ofP. caudatum. The results indicate that the infection of specific host cell compartments may depend on host genes, but not on different traits of the infecting symbiont species. The occurrence of killer and sensitive paramecia strains together in one pond is discussed with respect to the competitive advantage of the killer trait.

Molecular characterization of the D surface protein gene subfamily in Paramecium tetraurelia.

ABSTRACT. When Paramecium tetraurelia expresses the D serotype, detectable by serum tests, high molecular mRNA could be isolated, which corresponds to the molecular mass of the D surface protein. Using this D specific mRNA as a probe for screenings in different genomic libraries a subfamily of five very similar genes was found, named α‐51D, γ1‐51D, γ2‐51D, δ‐51D and ε‐51D. Each of them is about 8‐kb long, they show regions of identity to each other, and there is no evidence that any are defective genes or pseudogenes. Up to now serotype D is the only known serotype showing this phenomenon. Another novel feature is that two of the D isogenes are closely linked. The sequence for the entire coding region of the α‐51D gene has been determined, as well as the upstream and downstream noncoding regions. Its deduced amino acid sequence shows the same characteristic cysteine periodicity displayed by all other immobilization antigen (i‐ag) genes from Paramecium. However, in contrast to most other such genes, tandem repeats are missing from the 7599‐bp long coding region of the α‐51D gene. When the sequences of the type 51D genes are compared to each other, the similarity is very high and extends to coding as well as to noncoding regions. Similarity within noncoding regions is usually only observed for allelic i‐ag genes. We conclude that the type D genes constitute a family of isogenes that are nonallelic. They contain slightly different consensus sequences with possible functions as regulatory regions.

Characterization of Caedibacter endonucleobionts from the macronucleus of Paramecium caudatum and the identification of a mutant with blocked R-body synthesis

Cytology, DNA and host-symbiont relationships of x-like endosymbionts from Paramecium caudatum are described. The symbionts (Caedibacter caryophila, sp. nov.) live in the macronuclei of their hosts. They confer the killer trait upon their hosts and appear well adapted to their endonucleobiotic way of life. R bodies (proteinaceous ribbons associated with killing) are produced, but differ significantly from any of the four R-body classes previously described. C. caryophila and their R bodies were isolated. DNA was extracted from purified symbionts and used to demonstrate that one P. caudatum line harbors a natural mutant which is deficient in R-body production. Melting studies indicate a GC content of 34.6%. No sequence homology between the C. caryophila DNA and the coding sequence for type 51 R-body production was observed. C. caryophila is parasitic, causing the death of its hosts in starving cultures.