Magdalena Greczek-Stachura

Genetic analysis of the Paramecium aurelia species complex (Protozoa: Ciliophora) by classical and molecular methods

Abstract The Paramecium aurelia complex includes 15 species (sibling species) and is characterised by inbreeding (to varying degrees in different species), causing an increase in intra‐specific differentiation. Investigations into inter‐ and intraspecific differentiation of strains originating from remote habitats within species of the complex were carried out by classical inter‐strain crosses and molecular analyses (RAPD‐PCR fingerprints, ARDRA riboprints, RFLP‐PCR analysis). RAPD analysis showed that all species in the complex possessed characteristic band patterns and the majority were also polymorphic intra‐specifically. A correlation exists between the degree of inbreeding characteristic for a species with differentiation of DNAgenotypes revealed by RAPD analysis within species, where inbreeders showed substantial variability of band patterns and moderate inbreeders were highly similar. RFLP analysis (a 480bp fragment of the gene coding the Hsp 70 protein) with the application of restriction enzyme TruII distinguished among species, while digestion with restriction enzyme AluI distinguished groups of species (clusters) and both enzymes revealed intra‐species polymorphism within P. dodecaurelia. ARDRA riboprinting (using a fragment of SSU‐LSU rDNA, about 2400 bp) with restriction enzymes HhaI, AluI, HinfI, TaqI distinguished groups of species with different band patterns. The majority of enzymes also demonstrated intra‐specific differentiation within P. dodecaurelia. TaqI also revealed intraspecific differences in P. biaurelia and P. tetraurelia. All species in the P. aurelia complex showed a high percentage of surviving hybrid clones in F 1 obtained by conjugation and F2 obtained by autogamy in inter‐strain crosses. A low percentage was observed only in F2 inter‐strain hybrids of P. tredecaurelia, however no cytological changes in the nuclear apparatuses were detected and similar band patterns existed in the studied strains. Future studies, including sequencing of rDNA fragments, may disclose deeper relationships of the species.

New European stands of Paramecium pentaurelia, Paramecium septaurelia, and Paramecium dodecaurelia, genetic and molecular studies

New stands of rare species of the Paramecium aurelia complex were found in Europe, i.e. P. pentaurelia and P. dodecaurelia in Italy and P. septaurelia in Germany. The species were identified by mating reactions with the standard strains of each species. Their relationships with some other known strains of particular species were studied by classical strain crosses (survival in F1 and F2 generations) and by comparison of RAPD-PCR fingerprints. The presence of the cosmopolitan species P. tetraurelia in Italy was also recorded.

Seasonal and spatial variability of species occurrence of the Paramecium aurelia 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.

Polymorphism of Paramecium pentaurelia (Ciliophora, Oligohymenophorea) strains revealed by rDNA and mtDNA sequences.

Paramecium pentaurelia is one of 15 known sibling species of the Paramecium aurelia complex. It is recognized as a species showing no intra-specific differentiation on the basis of molecular fingerprint analyses, whereas the majority of other species are polymorphic. This study aimed at assessing genetic polymorphism within P. pentaurelia including new strains recently found in Poland (originating from two water bodies, different years, seasons, and clones of one strain) as well as strains collected from distant habitats (USA, Europe, Asia), and strains representing other species of the complex. We compared two DNA fragments: partial sequences (349 bp) of the LSU rDNA and partial sequences (618 bp) of cytochrome B gene. A correlation between the geographical origin of the strains and the genetic characteristics of their genotypes was not observed. Different genotypes were found in Kraków in two types of water bodies (Opatkowice-natural pond; Jordans Park-artificial pond). Haplotype diversity within a single water body was not recorded. Likewise, seasonal haplotype differences between the strains within the artificial water body, as well as differences between clones originating from one strain, were not detected. The clustering of some strains belonging to different species was observed in the phylogenies.

Polymorphism within Paramecium sexaurelia (Ciliophora, Oligohymenophorea) and description of a new stand of the species in China.

The presence of Paramecium sexaurelia from the Paramecium aurelia complex was recorded for the first time in China (Beijing). RAPD fingerprints (band patterns) of P. sexaurelia strains, the new strain from China and others from Asia, as well as from Europe and Puerto Rico, showed polymorphism within the species as several groups of genotypes characterized by different band patterns.

Molecular analysis [RAPD-PCR] of inter-strain hybrids of the Paramecium aurelia species complex [Ciliophora, Protozoa]

RAPD-PCR analysis showed that species of the Paramecium aurelia complex possessed characteristic band patterns and that the majority were also polymorphic intra-specifically. A comparison of band patterns was performed for some inter-strain hybrids within P. primaurelia, P. tetraurelia, P. pentaurelia, P. septaurelia, P. octaurelia, P. decaurelia, P. dodecaurelia, P. tredecaurelia, and P. quadecaurelia to band patterns characteristic for the parental strains. The investigations, however, did not reveal a close correlation between the degree of inbreeding characteristic for the species and similarity of genotypes. A low similarity of hybrid and parental band patterns was observed in P. octaurelia, P. dodecaurelia, P. quadecaurelia and also P. primaurelia. A high similarity of band patterns of hybrid and parental strains was found in P. tetraurelia, P. septaurelia, P. decaurelia, and P. tredecaurelia.

Strains of Paramecium decaurelia (Ciliophora, Protozoa) from Russia with molecular characteristics of other known strains of the species

The presence of Paramecium decaurelia from the Paramecium aurelia species complex was demonstrated in Yaroslavl, Russia, (European part, northwestern Russia) and in the Altai Mts (Asiatic part of Russia, western Siberia). RAPD-PCR fingerprints of the newly identified strains of P. decaurelia, rare throughout the world, were compared to those characteristic for the other known strains ofthe species. P. decaurelia strains show some polymorphism within species, strains from Russia have 60% similarity of band patterns, and strains from USA and Japan about 70% similarity of band patterns.

Photosynthetic efficiency of endosymbiotic algae of Paramecium bursaria originating from locations with cold and warm climates

Abstract Paramecium bursaria (Ciliophora) is a cosmopolitan unicellular organism that plays a significant role in aquatic ecosystems. P. bursaria contains symbiotic algae and this association is a mutual symbiosis. The aim of the present study was to determine the activity of photosystem II (PSII) in Chlorella sp. inside P. bursaria cells. Ciliates were incubated for 7 days at different temperatures from 6 to 18°C, under the circadian cycle: 12 h light/12 h dark, at light intensity of 200 μmol m-2 s-1 and under constant darkness conditions. The control group was kept at a temperature of 18°C under constant light conditions. Changes in PSII were monitored using different fluorescence parameters. Differences in responses between endosymbiotic algae of two P. bursaria strains – Ard7 from a warm climate and KD64 from a cold climate – were determined. The highest photosynthetic activity of P. bursaria green endosymbionts was observed at a temperature of 18°C, regardless of the light conditions. Algae from warm climate were more sensitive to cold temperature stress than algae from P. bursaria collected in cold climate.