Estimation of effective concentrations of ATP-regenerating enzymes in cilia of Paramecium caudatum.

The phosphoarginine shuttle system effectively regenerates ATP in the cilia of Paramecium caudatum. To estimate the effective concentration of ATP-regenerating enzymes, we attempted to reconstitute certain ATP-regenerating systems within the cilia of intact cortical sheets using exogenous enzymes and high-energy substances. The addition of phosphoenolpyruvate, which is one of the substrates in glycolysis, did not increase the ciliary beat frequency, whereas phosphocreatine together with exogenous creatine kinase, effectively increased the ciliary beat frequency. In the presence of 0.6 mg/ml creatine kinase and 0.4 mM phosphocreatine, the ciliary beat frequency was comparable to that produced by the addition of phosphoarginine. This result indicates that the reconstituted phosphocreatine shuttle system can work as an artificial ATP-regenerating system for ciliary movements. The effective concentration of creatine kinase in the reconstituted phosphocreatine shuttle system was estimated to be about 7.4 µM based on the molecular mass of creatine kinase (MW 81,000). Therefore, the effective concentration of arginine kinase in the cilia of live Paramecium is approximately 10 µM. This estimated concentration of intraciliary arginine kinase is sufficient to maintain a high ATP concentration throughout the cilia of P. caudatum.

Intraspecific genetic variation in Paramecium revealed by mitochondrial cytochrome C oxidase I sequences.

Studies of intraspecific genetic diversity of ciliates, such as population genetics and biogeography, are particularly hampered by the lack of suitable DNA markers. For example, sequences of the non-coding ribosomal internal transcribed spacer (ITS) regions are often too conserved for intraspecific analyses. We have therefore identified primers for the mitochondrial cytochrome c oxidase I (COI) gene and applied them for intraspecific investigations in Paramecium caudatum and Paramecium multimicronucleatum. Furthermore, we obtained sequences of the ITS regions from the same strains and carried out comparative sequence analyses of both data sets. The mitochondrial sequences revealed substantially higher variation in both Paramecium species, with intraspecific divergences up to 7% in P. caudatum and 9.5% in P. multimicronucleatum. Moreover, an initial survey of the population structure discovered different mitochondrial haplotypes of P. caudatum in one pond, thereby demonstrating the potential of this genetic marker for population genetic analyses. Our primers successfully amplified the COI gene of other Paramecium. This is the first report of intraspecific variation in free-living protozoans based on mitochondrial sequence data. Our results show that the high variation in mitochondrial DNA makes it a suitable marker for intraspecific and population genetic studies.